Search

This chapter examines how three secondary mathematics preservice teachers and two teacher educators rehearse and enact the core teaching practice of leading a whole-class discussion in a math methods course and in student teaching placements. Findings indicate that there was substantial variation in the three preservice teachers' opportunities to practice key aspects of leading a whole-class discussion, the type of feedback they received from the teacher educators, and the authenticity of the rehearsal. The opportunity to approximate practice and receive feedback played a significant role in the generative nature of the preservice teachers' enactments of a whole-class discussion in their student teaching placements.

Background: Affected patients exhibit alterations in psychomotor, intellectual, cognitive, emotional, behavioural and fine motor functions. Hepatic encephalopathy occurs as a complication of advance liver disease, either chronic or acute. Methods: 58 patients of hepatic encephalopathy was enrolled in our study which was done in Chhatrapati Shivagi Subharti hospital for the duration of 2 years to look for the correlation of grades of hepatic encephalopathy with serum ammonia levels. Results: In this study, the correlation of serum ammonia was significant with the grade of encephalopathy hence, the serum ammonia correlated well with the grade of encephalopathy for the patients during their hospital stay, suggesting that the serum ammonia level was an indicator of the clinical outcome of the patients during their hospital stay. Conclusions: In this study, we found that there is a strong correlation between serum ammonia level and severity of liver disease. Higher serum ammonia was found in severe liver disease. We found that High grade i.e. grade III and IV patients were having significantly raised ammonia level.

Arginase has been reported to reduce nitric oxide bioavailability in cardiovascular disease. However, its specific role in retinopathy has not been studied. In this study, we assessed the role of arginase in a mouse model of endotoxin-induced uveitis induced by lipopolysaccharide (LPS) treatment. Measurement of arginase expression and activity in the retina revealed a significant increase in arginase activity that was associated with increases in both mRNA and protein levels of arginase (Arg)1 but not Arg2. Immunofluorescence and flow cytometry confirmed this increase in Arg1, which was localized to glia and microglia. Arg1 expression and activity were also increased in cultured Muller cells and microglia treated with LPS. To test whether arginase has a role in the development of retinal inflammation, experiments were performed in mice deficient in one copy of the Arg1 gene and both copies of the Arg2 gene or in mice treated with a selective arginase inhibitor. These studies showed that LPS-induced increases in inflammatory protein production, leukostasis, retinal damage, signs of anterior uveitis, and uncoupling of nitric oxide synthase were blocked by either knockdown or inhibition of arginase. Furthermore, the LPS-induced increase in Arg1 expression was abrogated by blocking NADPH oxidase. In conclusion, these studies suggest that LPS-induced retinal inflammation in endotoxin-induced uveitis is mediated by NADPH oxidase-dependent increases in arginase activity.

DNA methylation alteration of several genes contributes to human tumorigenesis. Cyclin D2, a member of the D-type cyclins, is implicated in cell cycle regulation and malignant transformation. In our study, we examined the methylation status of the cyclin D2 promoter in small cell lung cancer (SCLC), nonsmall cell lung cancer (NSCLC), breast tumors and tumor cell lines. We observed that aberrant methylation of cyclin D2 was present in 32 of 56 (57%) SCLC cell lines, 7 of 32 (22%) SCLC tumor tissues; 25 of 61 (47%) NSCLC cell lines, 19 of 48 (40%) NSCLC tumor tissues; 18 of 30 (60%) breast tumor cell lines and 19 of 63 (30%) breast tumor tissues. Methylation was more frequent in the tumor cell lines compared to the primary breast and SCLC tumors (p = 0.007 and p = 0.001, respectively). Methylation was rare in the control tissue samples; 0 of 12 peripheral blood lymphocytes; 0 of 12 buccal epithelial cells; 0 of 18 nonmalignant lung tissues and 3 of 28 (11%) nonmalignant breast tissues. Promoter methylation correlated with loss of transcript by reverse transcription PCR (RT-PCR) in 9 of 11 (6 lung, 5 breast) tumor cell lines tested. Two cell lines that were not methylated also lacked expression, suggesting that other mechanisms of inactivation may be involved. Expression was restored by treatment with the demethylating agent, 5 aza 2' deoxycytidine, in all 9 methylated cell lines. Our results confirm earlier reports in breast cancer and indicate that aberrant methylation of cyclin D2 may contribute to the pathogenesis of the 2 major types of lung cancers.

An important method for silencing tumor suppressor genes in cancers is by aberrant methylation (referred to as methylation) of CpG islands in gene promoter regions. In lung cancer, methylation of the genes retinoic acid receptor beta-2 (RARbeta-2), CDH13 (H-cadherin), p16(INK4a) (p16), RASSF1A (RAS association domain family I) is frequent. Thus, we investigated methylation of these genes in 4 different types of specimens (oropharyngeal brushes, sputum samples, bronchial brushes and bronchioloalveolar lavage [BAL] samples) of the upper aerodigestive tract epithelium from heavy smokers without evidence of cancer but with morphometric evidence of sputum atypia and compared the frequencies of methylation in the different types of specimens. In addition, we also analyzed sputum samples from 30 never smokers for methylation of these genes. Our major findings are: (i) At least one gene was methylated in one or more specimens from 48% of the smokers. However, methylation was statistically significant less frequently in never smokers compared to smokers. (ii) In general, methylation occurred more frequently in samples from the central airways (sputum, bronchial brushes) compared to the peripheral airways (BAL) and only occasionally in the oropharynx. (iii) RARbeta-2 was the most frequently methylated gene, whereas the frequency of methylation for the other genes was lower. (iv) Data from sputum samples and bronchial brushes were comparable. Our findings suggest that detection of methylation should be investigated as an intermediate marker for lung cancer risk assessment and response to chemopreventive regimens.

Background:The mammary sentinel lymph node procedure can increase the detection of axillary metastases by 45% compared with standard axillary dissection. Some investigators have reported that reverse transcriptase-polymerase chain reaction (RT-PCR) increases metastasis detection even more, but it is uncertain whether a positive RT-PCR test in the face of a negative histological evaluation is clinically meaningful. Methods:RT-PCR for epithelial glycoprotein 2 and cytokeratin 19 was performed on sentinel and pooled nonsentinel axillary lymph nodes from 108 women with clinical stage I or II breast cancer who were followed up for a median of 40 months. Results:Axillary metastases were detected on standard tissue sections in 26% and by RT-PCR in 30%. Results for the two tests were concordant for 80% of the cases. RT-PCR upstaged 16%. Tumors from women whose lymph nodes were positive only by RT-PCR were phenotypically similar to those from women with no metastases detected by any method. Moreover, 4-year actuarial distant disease-free survival was 100% for women with metastases detected by RT-PCR only, as compared with 74% for those with metastases detected by routine histology (P = .03) and 93% for those with no metastases detected by either method (P = .04). Conclusions:Analysis of sentinel lymph nodes by RT-PCR for epithelial glycoprotein 2 and cytokeratin 19 is unlikely to provide clinically useful information.

Aberrant methylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of lung cancers. There are major smoke exposure, histology, geography and gender-related changes in non-small cell lung cancer (NSCLC). We investigated smoking-related, histologic, geographic and gender differences in the methylation profiles of resected NSCLCs. We examined 514 cases of NSCLC and 84 corresponding nonmalignant lung tissues from 4 countries (USA, Australia, Japan and Taiwan) for the methylation status of 7 genes known to be frequently methylated in lung cancers [p16, RASSF1A (RAS association domain family 1), APC, RARbeta, CDH13, MGMT and GSTP1]. Multivariate analyses were used for data analysis. Adenocarcinoma was the major histologic type in women and never smokers; analyses that involved smoke exposure and gender were limited to this histology. Our major findings are a) methylation status of any single gene was largely independent of methylation status of other genes; b) the rates of methylation of p16 and APC and the mean Methylation Index (MI), a reflection of the overall methylation status, were significantly higher in ever smokers than in never smokers; c) the mean MI of tumors arising in former smokers was significantly lower than the mean of current smokers; d) the methylation rates of APC, CDH13 and RARbeta were significantly higher in adenocarcinomas than in squamous cell carcinomas; e) methylation rates of MGMT and GSTP1 were significantly higher in the USA and Australian cases than in those from Japan and Taiwan; and (f) no significant gender-related differences in methylation patterns were noted. Our findings demonstrate important smoke exposure, histologic type and geography-related differences in the methylation profiles of NSCLC tumors.

Somatic cells express genes that suppress telomerase activity and these genes may be inactivated in tumour cells. We postulated that cancer cells acquire immortality by activation of telomerase by the loss of such a gene. We have reported recently that a telomerase repressor gene may be located on 10p15.1 by deletion mapping using microcell-mediated chromosome transfer (MMCT), radiated microcell fusion (RMF), fluorescent in situ hybridization (FISH) and STS analysis. To independently confirm this result, we correlated expression of RNA component of telomerase (hTR) as a marker of telomerase expression by in situ hybridization with allelic loss in pulmonary carcinoid tumours. Unlike most malignant tumours, pulmonary carcinoids (which are low-grade malignant tumours) are heterogeneous for telomerase expression. Loss of 5 closely spaced polymorphic markers on 10p15.1, especially D10S1728, were highly correlated with hTR expression. In an additional experiment, 10p15.1 showed higher and more significant correlation than any region of 3p where it has been predicted as another chromosomal location of telomerase repressor with allelic loss of the region. Our findings strongly suggest that 10p15.1 harbours a gene involved in repression of telomerase RNA component in human somatic cells and each putative repressor (on 3p and 10p) may act independently. © 2001 Cancer Research Campaign http://www.bjcancer.com

Background: The recently identified RASSF1 locus is located within a 120-kilobase region of chromosome 3p21.3 that frequently undergoes allele loss in lung and breast cancers. We explored the hypothesis that RASSF1 encodes a tumor suppressor gene for lung and breast cancers. Methods: We assessed expression of two RASSF1 gene products, RASSF1A and RASSF1C, and the methylation status of their respective promoters in 27 non-small-cell lung cancer (NSCLC) cell lines, in 107 resected NSCLCs, in 47 small-cell lung cancer (SCLC) cell lines, in 22 breast cancer cell lines, in 39 resected breast cancers, in 104 nonmalignant lung samples, and in three breast and lung epithelial cultures. We also transfected a lung cancer cell line that lacks RASSF1A expression with vectors containing RASSF1A complementary DNA to determine whether exogenous expression of RASSF1A would affect in vitro growth and in vivo tumorigenicity of this cell line. All statistical tests were two-sided. Results: RASSF1A messenger RNA was expressed in nonmalignant epithelial cultures but not in 100% of the SCLC, in 65% of the NSCLC, or in 60% of the breast cancer lines. By contrast, RASSF1C was expressed in all nonmalignant cell cultures and in nearly all cancer cell lines. RASSF1A promoter hypermethylation was detected in 100% of SCLC, in 63% of NSCLC, in 64% of breast cancer lines, in 30% of primary NSCLCs, and in 49% of primary breast tumors but in none of the nonmalignant lung tissues. RASSF1A promoter hypermethylation in resected NSCLCs was associated with impaired patient survival (P = .046). Exogenous expression of RASSF1A in a cell line lacking expression decreased in vitro colony formation and in vivo tumorigenicity. Conclusion: RASSF1A is a potential tumor suppressor gene that undergoes epigenetic inactivation in lung and breast cancers through hypermethylation of its promoter region.

Monozygotic twins, each of whom has breast cancer, offer a natural study population for gene-environmental interactions as causation of cancer, because they are genetically identical. If heritable factors play a large role in the origin of a neoplasm, disease concordance should be significant in monozygotic twins. Two monozygotic triplet sisters carrying a germline BRCA1 gene mutation (5382insC) who both developed breast cancer at early ages were studied for loss of heterozygosity (LOH) in their microdissected, paraffin-embedded tumors along with control blood and stromal breast tissue at 19 chromosomal arms using 161 microsatellite markers. Microdissected areas of normal lobular and ductal epithelium and ductal in situ carcinoma were also studied for LOH using a subset of microsatellite markers. The mother's DNA (extracted from peripheral blood lymphocytes) was analyzed to determine the parental allele under LOH in each case. Both tumors demonstrated similar histologic features suggestive of a secretory variant of ductal carcinoma. The tumors from both sisters had similar overall LOH frequency expressed by the fractional allelic loss (FAL) indices (0.56 vs. 0.60) and demonstrated concordance for loss or retention at 82 of 97 informative markers (85% correlation). In addition, detailed mapping analysis of several chromosomal arms revealed that identical breakpoints were detected in both tumors at several chromosome regions. Finally, in both sisters' tumors, when a chromosome exhibited allelic loss, all of the markers exhibited LOH of the same parental allele even when there were intervening regions of retention of heterozygosity. In contrast, 17 archival sporadic breast carcinomas demonstrated a wide range of FAL indexes and highly individual patterns of LOH. Our findings support the hypothesis that inherited factors play a role in the development of the multiple somatic deletions occurring in breast carcinomas. Whether one of these factors is the mutant BRCA1 allele or some other gene(s) remains to be determined. Genes Chromosomes Cancer 28:359-369, 2000.

Background: Several molecular changes, including loss of heterozygosity (i.e., deletion of one copy of allelic DNA sequences) and alterations in microsatellite DNA, have been detected early in the pathogenesis of lung cancer, even in histologically normal epithelium. In the bronchial epithelium of patients with lung cancer, we have determined the frequency, size, and patterns of molecularly abnormal clonal patches. Methods: We studied formalin-fixed, paraffin-embedded samples from 16 surgically resected lung carcinomas (five squamous cell carcinomas, four small-cell carcinomas, six adenocarcinomas, and one large-cell carcinoma). From each carcinoma, we microdissected foci (each containing about 200 cells) of tumor tissue and equivalent samples of histologically normal and abnormal epithelium. Furthermore, multiple discontinuous foci of bronchial epithelium were analyzed from methanol-fixed samples from three additional patients with lung cancer (two with squamous cell carcinoma and one with adenocarcinoma). We used two-step polymerase chain reaction-based assays involving 12 microsatellite markers at seven chromosomal regions frequently deleted in lung cancer. Results: Two hundred eighteen foci of nonmalignant bronchial epithelium (195 of histologically normal or slightly abnormal epithelium and 23 of dysplastic epithelium) were studied from the 19 surgically resected lobectomy specimens. Thirteen (68%) of the 19 specimens had at least one focus of bronchial epithelium with molecular changes. At least one molecular abnormality was detected in 32% of the 195 histologically normal or slightly abnormal foci and in 52% of the 23 dysplastic foci. Extrapolating from our two-dimensional analyses, we estimate that most clonal patches contain approximately 90000 cells. Although, in a given individual, tumors appeared homogeneous with respect to molecular changes, the clonally altered patches of mildly abnormal epithelium were heterogeneous. Conclusions: Our findings indicate that multiple small clonal or subclonal patches containing molecular abnormalities are present in normal or slightly abnormal bronchial epithelium of patients with lung cancer.

Mucins comprise an important class of tumor-associated antigens. The objectives of the present study were (a) to establish an in vitro model system using human non-small cell lung adenocarcinoma cell lines NCIH650 and NCIH2077 (b) provide evidence that these cell lines secrete mucin in culture conditions and (c) investigate the effects of select secretagogues on mucin secretion. The cell lines were established in ACL-4 medium containing several growth factors and retinoic acid and 5% fetal calf serum. The high molecular weight glycoconjugates secreted in the culture medium were purified by ammonium sulfate precipitation and Superose 6 and Superose 12 FPLC chromatography. The purified high molecular weight glycoconjugate fraction and the carcinoma cells were shown to have mucin by dot blot, Western blot and immunohistochemical analysis, respectively, using specific antibodies to purified major mucin, HTM-1. Also, incorporation experiments with mucin precursor 3H-glucosamine demonstrated that the cells indeed synthesize high molecular weight mucins. The effects of secretagogues such as, 8-bromocyclic AMP, ionomycin, phorbol-12-myristate-13-acetate and neutrophil elastase on mucin secretion were also investigated. Only 8-bromocyclic AMP and neutrophil elastase influenced mucin secretion. These studies provided strong evidence that the lung adenocarcinoma cell lines secrete high molecular weight mucins in culture conditions and only two of the four tested secretagogues significantly increased mucin secretion. Thus, this in vitro model system may be useful in determining alterations in mucin structure, if any, in lung adenocarcinomas as well as in studying the regulation of mucin gene expression.

11q23-24 chromosome is a region containing frequent allelic loss (loss of heterozygosity; LOH) in human cancers. To examine cancer-related allelic loss in the region between D11S940 and APOC3, we used 17 polymorphic markers and allotyped 28 lung cancer-derived cell lines and their corresponding matched lymphoblastoid cell lines. LOH was found in 71.4% (20/28) of the lung cancer cell lines and was localized to two distinct minimal regions of loss. One region is bracketed by markers D11S1647 and NCAM2 and contains the gene encoding the beta isoform of the A subunit of the human protein phosphatase 2A (PPP2R1B). Recently, mutations in this gene were described in lung and colon cancers, suggesting that PPP2R1B functions as a tumor-suppressor gene. A second minimal region of loss was defined between markers D11S1792 and D11S1885, a region estimated to be less than I Mb. Thus, chromosome 11 likely harbors two sites of suppressor oncogene activity in lung cancer, one defined by the PPP2R1B gene and the second located telomeric to PPP2R1B. This study facilitates the identification and cloning of a second critical tumor-suppressor gene involved in lung cancer, and possibly a variety of other cancers, on human chromosome band 11q23.

BACKGROUND Recent classifications identify four categories of neuroendocrine (NE) tumors of the lung: low grade typical carcinoid (TC), intermediate grade atypical carcinoid (AC), and high grade large cell neuroendocrine carcinoma (LCNEC) and small cell lung carcinoma (SCLC). METHODS The authors studied the molecular changes present in 59 archival NE tumors (10 TCs, 11 ACs, 18 LNECs, and 20 SCLCs). Utilizing microdissection and polymerase chain reaction-based assays, the authors examined loss of heterozygosity (LOH) at ten chromosomal regions frequently deleted in lung tumors (3p, 5q, 11q, 13q, and 17 p) and for mutations at the p53 and ras genes. RESULTS With the exception of ras gene mutations, the majority of these changes frequently were present in carcinomas and were present at lower frequencies in carcinoids. LOH at one or more 3p regions was the most frequent change found in the carcinoids. A relatively high incidence of LOH at the MEN1 gene was common in all NE lung tumors. The incidence of LOH and p53 gene abnormalities progressively increased with increasing severity of tumor type. The patterns of p53 gene mutations were different between AC and high grade NE tumors. LOH at 5q21 was correlated with poor survival in the carcinoid group. CONCLUSIONS Although NE lung tumors have varied etiologies, the results of the current study support the clinicopathologic concept that they represent a spectrum ranging from low grade TC to the highly malignant NE carcinomas. Cancer 1999;85:600–7. © 1999 American Cancer Society.

The goal of our study was to develop a panel of tumor cell lines along with paired non-malignant cell lines or strains collected from breast cancers, predominantly primary tumors. From a total of 189 breast tumor samples consisting of 177 primary tumors and 12 metastatic tissues, we established 21 human breast tumor cell lines that included 18 cell lines derived from primary tumors and 3 derived from metastatic lesions. Cell lines included those from patients with germline BRCA1 and FHIT gene mutations and others with possible genetic predisposition. For 19 tumor cell lines, we also established one or more corresponding non-malignant cell strains or B lymphoblastoid (BL) lines, which included 16 BL lines and 7 breast epithelial (2) or stromal (5) cell strains. The present report describes clinical, pathological and molecular information regarding the normal and tumor tissue sources along with relevant personal information and familial medical history. Analysis of the breast tumor cell lines indicated that most of the cell lines had the following features: they were derived from large tumors with or without axillary node metastases; were aneuploid and exhibited a moderate to poorly differentiated phenotype; were estrogen receptor (ER)- and progesterone receptor (PR)-negative; and overexpressed p53 and HER2/neu proteins. Of 13 patients with primary breast cancers receiving curative intent mastectomies, 7 were dead after a mean period of 10 months. Our panel of paired tumor and non-malignant cell lines should provide important new reagents for breast cancer research.

Where and how frequently p53 abnormalities are involved in the development of pleomorphic adenoma (PA) and its malignant progression to carcinoma was investigated. The presence of p53 gene abnormalities was analyzed in eight patients with carcinoma in pleomorphic adenoma (CPA) by polymerase chain reaction (PCR)-based assays and immunohistochemistry. Normal salivary gland tissue, adenomatous, transitional and carcinomatous areas were microdissected from archival microslides and analyzed for allelic deletions of the p53 gene using two microsatellite markers at the p53 locus; dinucleotide (CA)n repeat and pentanucleotide (AAAAT)n repeat. Loss of heterozygosity (LOH) of the p53 gene was detected in 5796 of adenomas, 86% of transitional lesions and 86% of carcinomas. In contrast, overexpression of p53 oncoprotein was noted immunohistochemically in 13% of adenomas, 50% of transitional areas and 75% of carcinomas. All of the tumors with immunoreactivity for p53 oncoprotein demonstrated LOH. Moreover, when LOH was present in adenomatous or transitional areas, the identical LOH was always detected in the corresponding carcinomatous areas in the same CPA tumors. These findings indicate that p53 gene mutation is an early event and occurs frequently at an early stage of precancerous lesions and may be responsible for most cases of malignant transformation of PA.

Abnormalities of the p53 tumor suppressor gene were investigated in 22 foci from 14 adenoid cystic carcinomas (ACC) by polymerase chain reaction (PCR)-based assays for dinucleotide (CA)n and pentanucleotide (AAAAT)n repeat polymorphisms and by immunohistochemical staining for oncoprotein expression. Adenoid cystic carcinomas were divided into lower grade (tubular and cribriform) subtypes and higher grade (trabecular and solid) subtypes. Histologically identified tumor cells were precisely microdissected from archival microslides and were used for molecular analysis. The overall frequency of p53 gene mutations detected by PCR-loss-of-heterozygosity (LOH) analysis was 57% and was higher than the frequency of over-expression of p53 oncoprotein detected by immunostaining (43%). In the molecular analysis of individual histological subtype foci, the number of foci with p53 gene mutation was significantly greater in the higher grade subtype foci than in the lower grade subtype foci and was greatest in solid-type foci (100%). In all six tumors in which histologically different foci were present in the same tumors, mutations of the p53 gene were detected. When tumor heterogeneity of the p53 gene was present among different histological foci in the same tumors, the mutations were always detected in the higher grade foci. When lower and higher grade foci were present in the same tumors, the identical mutations detected in the lower grade foci were present in the corresponding higher grade foci. These findings indicate that abnormalities of the p53 gene are involved in carcinogenesis and/or progression of this tumor and, furthermore, suggest that molecular analyses of ACC may provide information of prognostic importance.

Allelic loss is a hallmark of tumor suppressor gene (TSG) inactivation. We have allelotyped 29 paired lymphoblastoid and lung cancer cell lines derived from 11 patients with small cell (SCLC) and 18 patients with non-small cell lung carcinomas (NSCLC). Statistical analysis indicated that a threshold of 30% separated non-random allelic loss from the random genetic deletions of malignancy. We have identified non-random allelic loss at 42 of 54 (78%) specific chromosomal regions examined, with 22 regions (52%) common between the two major lung cancer histologic types. There were 3 regions (7%) with allelic loss specific for SCLC and 17 regions (41%) specific for NSCLC. Furthermore, there were significant differences in loss of heterozygosity (LOH) frequencies between NSCLC and SCLC at 13 regions on eight chromosome arms (3p, 5q, 6q, 9p, 10q, 11p, 13q, and 19p). Eight homozygous deletions were present in seven cell lines at four regions, 3p12, 3p14.2, 9p21, and 10q23–25. We have also identified novel sites of chromosomal deletions. In particular, there was frequent loss at 11p13 in SCLC and loss at 6p21.3 and 13q12.3 in NSCLC. In this study, we demonstrate that a) non-random allelic losses in lung cancer involve multiple regions; b) some losses are common to both NSCLC and SCLC subtypes, whereas others are subtype specific; c) there are genetic deletions at novel chromosomal regions; and d) several homozygous deletions have been noted. Our studies demonstrate the usefulness of continuous cell lines for detailed allelotyping, for comparing genetic abnormalities between SCLC and NSCLC, and for identifying homozygous deletions. Genes Chromosomes Cancer 21:308–319, 1998. © 1998 Wiley-Liss, Inc.

Background: Most lung cancers are attributed to smoking. These cancers have been associated with multiple genetic alterations and with the presence of preneoplastic bronchial lesions. In view of such associations, we evaluated the status of specific chromosomal loci in histologically normal and abnormal bronchial biopsy specimens from current and former smokers and specimens from nonsmokers. Methods: Multiple biopsy specimens were obtained from 18 current smokers, 24 former smokers, and 21 nonsmokers. Polymerase chain reaction-based assays involving 15 polymorphic microsatellite DNA markers were used to examine eight chromosomal regions for genetic changes (loss of heterozygosity [LOH] and microsatellite alterations). Results: LOH and microsatellite alterations were observed in biopsy specimens from both current and former smokers, but no statistically significant differences were observed between the two groups. Among individuals with a history of smoking, 86% demonstrated LOH in one or more biopsy specimens, and 24% showed LOH in all biopsy specimens. About half of the histologically normal specimens from smokers showed LOH, but the frequency of LOH and the severity of histologic change did not correspond until the carcinoma in situ stage. A subset of biopsy specimens from smokers that exhibited either normal or preneoplastic histology showed LOH at multiple chromosomal sites, a phenomenon frequently observed in carcinoma in situ and invasive cancer. LOH on chromosomes 3p and 9p was more frequent than LOH on chromosomes 5q, 17p (17p13; TP53 gene), and 13q (13q14; retinoblastoma gene). Microsatellite alterations were detected in 64% of the smokers. No genetic alterations were detected in nonsmokers. Conclusions: Genetic changes similar to those found in lung cancers can be detected in the nonmalignant bronchial epithelium of current and former smokers and may persist for many years after smoking cessation.

Three hundred and five cases of high altitude pulmonary oedema (HAPO) hospitalised in eastern Himalayan region have been analyzed. Incidence of HAPO was 5.5 per cent. Eighty per cent cases occurred during latter half of the year. Fifty six per cent of cases belonged to the third decade of life. HAPO cases occurred most commonly between the height of 2740 m to 5960 m. Eighty three per cent cases developed symptoms within 72 hours of induction to high altitude and 65.9 per cent suffered from the illness despite complete acclimatization. Breathlessness, headache and cough were the commonest symptoms. Tachycardia and tachypnoea was present in all cases. Twenty five per cent cases showed various ECG abnormalities. Mortality rate was 0.98 per cent.

The escape of tumor cells from immune recognition is a central problem in tumor immunology. Here, we examined the functional role of somatic beta 2-microglobulin (beta2m) gene mutations in human lung and breast cancers. Using single-strand conformational polymorphism (SSCP) analysis and DNA sequencing, we found mutations in the beta2m gene in 2 of 110 tested lung, colon and breast tumors and tumor cell lines. No mutations were identified in 63 breast cancer tumors, in B-lymphoblastoid cell lines or normal tissues from these or other patients. In these cell lines, beta2m protein was undetectable by Western blot analysis and there was no MHC class I on their cell surface even after treatment with interferon-gamma. Transfection of these tumor cell lines with the beta2m gene, but not addition of purified beta2m protein restored MHC expression without addition of exogenous pepticles, indicating that endogenous beta2m expression is necessary for proper intracellular MHC assembly and stabilization by endogeneous pepticles. Mutation in beta2m caused cell line H2009 to be resistant to specific lysis by influenza virus-specific CTL from HLA matched donors, and transfection of the beta2m gene restored this killing. A small cell lung cancer cell line with low class I expression and with a normal beta2m genomic sequence nonetheless also demonstrated increased class I expression after transfection of the beta2m expression vector alone, indicating that the availability of beta2m may be rate limiting for MHC assembly in this line. Our results indicate that somatic mutations or selective loss of expression of the beta2m gene in human lung cancer is rare, but can cause defective MHC class I expression and function allowing these cells to escape recognition by cytotoxic T cells.

Background : Carcinogenesis is a multistep process, which may begin as a consequence of chromosomal changes. Deletions in the short arm of chromosome 9 (9p) have been observed in lung carcinomas. In addition, morphologically recognizable preneoplastic lesions, frequently multiple in number, precede onset of invasive carcinomas. Purpose : We tested for deletions and loss of heterozygosity (LOH) at 9p loci in preneoplastic and neoplastic foci in lungs of patients with non-small-cell lung carcinomas (NSCLCs). Methods : Seven archival, paraffin-embedded, surgically resected NSCLC specimens were selected. They were predominantly from patients with adenocarcinomas and contained multiple preneoplastic lesions, including hyperplasia, metaplasia, dysplasia, and carcinoma in situ (CIS). Fifty-three histologically identified preneoplastic and malignant lesions present in bronchi, bronchioles, and alveoli were precisely microdissected from stained tissue sections with a micromanipulator. Stromal lymphocytes were used to determine constitutional heterozygosity. The specimens were analyzed for LOH using polymerase chain reaction-based assays for polymorphism in dinucleotide repeats (microsatellite markers) in interferon alfa (IFNA) and D9S171 loci on 9p. Results : All seven cases were constitutionally heterozygous for one or both microsatellite markers. Five of seven cases had LOH at one or both 9p loci in the invasive primary cancers (doubly informative cases). Four of these five cases also revealed LOH in preneoplastic foci. In the doubly informative cases, LOH was detected in five (38%) of 13 foci of hyperplasia, four (80%) of five foci of dysplasia, and three (100%) of three CIS lesions. LOH was detected in preneoplastic lesions from all regions of the respiratory tract, including bronchi, bronchioles, and alveoli, and involved five different cell types. The identical allele was lost from both the preneoplastic lesions and the corresponding tumors (12 of 12 lesions, 17 of 17 comparisons), a phenomenon we have referred to as allele-specific mutation. Statistical analyses employing a cumulative binomial test demonstrated that the probabilities of such findings occurring by chance are 2.4 x 10 -4 and 7.6 x 10 -6 , respectively. From comparisons with the previously published data on other chromosomal abnormalities in the same tissue specimens, it appears that LOH at 3p and 9p loci occurred early in the hyperplasia stage, but the ras gene point mutations were relatively late, at the CIS stage. Conclusions: LOH at 9p loci occurs at the earliest stage in the pathogenesis of lung cancer and involves all regions of the respiratory tract. LOH in NSCLC is not random but targets a specific allele in individuals. Studying preneoplastic lesions may help identify intermediate markers for risk assessment and chemoprevention.

A high-molecular-weight mucin (Mr approximately 11.0 x 10(6)) was purified from canine tracheal pouch secretions. The mucin was deglycosylated by treatment with trifluoromethane sulfonic acid for 8 h at 8 degrees C and subsequently with alpha-N-acetylgalactosaminidase. These treatments almost completely removed the carbohydrate moieties. The amino acid compositions of the deglycosylated and native mucins were similar, indicating that the deglycosylation procedure used did not cause notable degradation of the protein core. Antiserum specific for deglycosylated canine tracheal mucin was produced by immunization of rabbit with the antigen. RNA was isolated from fresh canine tracheal epithelial cells by extraction with guanidine isothiocyanate/hydrochloride and further fractionated by chromatography on oligo(dT)-cellulose to yield poly(A)+ RNA. The poly(A)+ RNA was translated in a rabbit reticulocyte cell-free translation system using [35S]methionine and [3H]leucine as radiolabels. The translation products were analyzed by gel electrophoresis and fluorography before and after immunoprecipitation with the antiserum to deglycosylated mucin. A labeled product of molecular weight 72,000 was present in the immunoprecipitate. When canine liver poly(A)+ RNA was used as control, no radioactivity above background was detected in the immunoprecipitate. It is concluded that the primary translation product of the canine tracheal epithelial cells is a 72,000-D protein and the monomer subunit of the mucin is about 167,000 D. Thus, in the native state, the canine tracheal mucin consists of several associating subunits.

Two high-Mr mucus glycoproteins (mucins), CTM-A and CTM-B, were highly purified from canine tracheal pouch secretions, and their macromolecular properties as well as polymeric structure were investigated. On SDS/composite-gel electrophoresis, a diffuse band was observed for each mucin. Polyacrylamide-gel electrophoresis using 6% gels also showed the absence of low-Mr contaminants in the mucins. Comparison of chemical and amino acid compositions revealed significant differences between the two mucins. Using a static-laser-light-scattering technique, CTM-A and CTM-B were found to have weight-average Mr values of about 11.0 x 10(6) and 1.4 x 10(6) respectively. Both mucins showed concentration-dependent aggregation in buffer containing 6 M-guanidine hydrochloride. Under similar experimental conditions, reduced-alkylated CTM-A had an Mr of 5.48 x 10(6) and showed no concentration-dependent aggregation. Hydrophobic properties of the mucins, investigated by the fluorescent probe technique using mansylphenylalanine as the probe, showed the presence of a large number of low-affinity (KD approx. 10(5) M) binding sites. These sites appeared to be located on the non-glycosylated regions of the protein core, since Pronase digestion of the mucins almost completely eliminated probe binding. Reduction of disulphide bonds of CTM-A and CTM-B did not significantly alter the probe-binding properties. Also, addition of increasing NaCl concentrations (0.03-1.0 M) to the buffer caused only a small change in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable degradation, using a combination of chemical and enzymic methods. On SDS/PAGE the protein core was estimated to have an Mr of approx. 60,000. On the basis of the protein and carbohydrate contents of the major mucin CTM-A, the mucin monomer was calculated to have an Mr of approx. 140,000. The high Mr (11 x 10(6] observed by physical methods is therefore due to self-association of the mucin monomer subunits.

Background We attempted to clone candidate genes on 10p14–15 which may regulate hTERT expression, through exon trapping using 3 BAC clones covering the region. After obtaining 20 exons, we examined the function of RGM249 (RGM: RNA gene for miRNAs) we cloned from primary cultured human hepatocytes and hepatoma cell lines. We confirmed approximately 20 bp products digested by Dicer, and investigated the function of this cloned gene and its involvement in hTERT expression by transfecting the hepatoma cell lines with full-length dsRNA, gene-specific designed siRNA, and shRNA-generating plasmid. Results RGM249 showed cancer-dominant intense expression similar to hTERT in cancer cell lines, whereas very weak expression was evident in human primary hepatocytes without telomerase activity. This gene was predicted to be a noncoding precursor RNA gene. Interestingly, RGM249 dsRNA, siRNA, and shRNA inhibited more than 80% of hTERT mRNA expression. In contrast, primary cultured cells overexpressing the gene showed no significant change in hTERT mRNA expression; the overexpression of the gene strongly suppressed hTERT mRNA in poorly differentiated cells. Conclusion These findings indicate that RGM249 might be a microRNA precursor gene involved in the differentiation and function upstream of hTERT.

The ras family consists of three evolutionarily conserved genes, H- ras , N- ras , and K- ras . The ras genes code for 21-kDa proteins that mediate signal transduction between cell surface receptors and intracellular regulatory molecules. The proteins attach to the inner surface of the cytoplasmic membrane via a posttranslationally added farnesyl group. This lipid modification is essential for function, and considerable recent interest has focused on agents that inhibit the enzyme responsible, farnesyl transferase, as a mechanism to block the growth of tumors having ras mutations (1). Ras is homologous to a larger family of “G proteins” that exist in two states, GDP- and GTP-bound. Only the GTP-bound form is effective at mediating growth response, and there is a dynamic interconversion between the two forms. Point mutations of ras are the most frequent dominant oncogene mutation found in human tumors and usually target only three codons: 12, 13, and 61. Most activating mutations are defective in GTPase activity and thus are locked into the GTP-bound form, resulting in continuous growth stimulation. Detection of ras mutations in tumors enables us to understand cancer biology and pathogenesis and may be of clinical importance by providing information useful for early diagnosis and prognosis and the development of novel therapeutic approaches (1)(2). As discussed below, ras mutations may be detected by a large variety of methodologies, both conventional and, as reported in this issue of Clinical Chemistry , supersensitive (3). Although about 20–25% of human tumors have ras mutations, especially involving the K- ras gene, the distribution demonstrates considerable tumor type specificity (4)(5). Thus, although K- ras mutations are found in nearly 90% of pancreatic carcinomas and about 50% of colorectal carcinomas, they are rare in breast cancers. Lung cancers are an example of the heterogeneity …

An important method for silencing tumor suppressor genes in cancers is by aberrant methylation (referred to as methylation) of CpG islands in gene promoter regions. In lung cancer, methylation of the genes retinoic acid receptor beta-2 (RARbeta-2), CDH13 (H-cadherin), p16(INK4a) (p16), RASSF1A (RAS association domain family I) is frequent. Thus, we investigated methylation of these genes in 4 different types of specimens (oropharyngeal brushes, sputum samples, bronchial brushes and bronchioloalveolar lavage [BAL] samples) of the upper aerodigestive tract epithelium from heavy smokers without evidence of cancer but with morphometric evidence of sputum atypia and compared the frequencies of methylation in the different types of specimens. In addition, we also analyzed sputum samples from 30 never smokers for methylation of these genes. Our major findings are: (i) At least one gene was methylated in one or more specimens from 48% of the smokers. However, methylation was statistically significant less frequently in never smokers compared to smokers. (ii) In general, methylation occurred more frequently in samples from the central airways (sputum, bronchial brushes) compared to the peripheral airways (BAL) and only occasionally in the oropharynx. (iii) RARbeta-2 was the most frequently methylated gene, whereas the frequency of methylation for the other genes was lower. (iv) Data from sputum samples and bronchial brushes were comparable. Our findings suggest that detection of methylation should be investigated as an intermediate marker for lung cancer risk assessment and response to chemopreventive regimens.

To determine the role of methylation of HIC1, a candidate tumor suppressor gene on 17p13.3, in various types of pediatric tumors.We examined the methylation status of the HIC1 promoter by methylation specific PCR in 157 pediatric tumors and 27 nonmalignant tissues. We correlated methylation with mRNA expression by reverse transcription-PCR in eight tumor-derived cell lines.HIC1 methylation was frequent in medulloblastomas (80%, 12 of 15), retinoblastomas (67%, 6 of 9), rhabdomyosarcomas (59%, 13 of 22), germ cell tumors (55%, 6 of 11), and neurouroblastic tumors (36%, 14 of 39); neuroblastomas (43%, 12 of 28); ganglioneuromas (17%, 1 of 6); and ganglioneuroblastomas (20%, 1 of 5). In contrast, a low incidence of methylation was observed in osteosarcomas (17%, 2 of 12), Ewing's tumors (9%, 1 of 11), Wilms' tumors (3%, 1 of 31), and hepatoblastomas (0%, 0 of 7). HIC1 methylation was more frequent in the aggressive alveolar subtype of rhabdomyosarcomas (100%, 8 of 8) than the embryonal subtype (33%, 4 of 12; P0.005) and was rare in the nonmalignant tissues examined. Methylation was also demonstrated by sequencing in nine randomly selected tumor samples. Seven of eight pediatric tumor cell lines examined were methylated and showed loss or reduced HIC1 mRNA. Expression was strongly induced in all cell lines by treatment with the demethylating agent 5-aza 2'deoxycytidine.Our data suggest that aberrant methylation of HIC1 may play a role in the pathogenesis of specific pediatric tumors.

TMS1 (target of methylation-induced silencing) is a CpG island-associated gene that functions in the regulation of apoptosis and encodes a caspase recruitment domain, a recently described motif found in apoptotic signaling molecules. Recent evidence suggests that silencing of genes in the apoptotic pathway contribute to human carcinogenesis. We examined the DNA methylation status of the TMS1 promoter in lung and breast tumor tissues, tumor cell lines and nonmalignant tissues by methylation-specific polymerase chain reaction (MSP) and its mRNA expression by reverse transcription PCR. Aberrant methylation of TMS1 was present in 70% (40 of 57) of small cell lung cancer (SCLC) cell lines and 41% (13 of 32) of SCLC tumor tissues, 48% (29 of 61) of non small cell lung cancer (NSCLC) cell lines and 40% (28 of 70) of NSCLC tumor tissues and 46% (12 of 26) of breast cancer cell lines and 32% (20 of 63) of breast tumor tissues. Methylation was absent in the peripheral blood lymphocytes and buccal epithelium from healthy volunteers, as well as in nonmalignant lung tissues and was rare in nonmalignant breast tissues 7% (2 of 30). DNA methylation was confirmed by sequence analysis and the methylation status correlated inversely with TMS1 RNA expression in 18 cell lines tested. RNA expression was restored by treatment with the demethylating agent 5-aza-2′-deoxycytidine, in 4 of 4 methylated cell lines that lacked the TMS1 transcript. Our results suggest that methylation of TMS1 may play a role in the pathogenesis of small cell and non small lung and breast cancers. © 2003 Wiley-Liss, Inc.

Chfr, a mitotic stress checkpoint gene, regulates a prophase delay in cells exposed to agents that disrupt microtubules, such as nocodazole and taxol. In the present study, we report that Chfr is frequently methylated in cell lines derived from tumors of the colon (80%), brain (100%) and bone (100%). In addition, Chfr was methylated in 37% of primary colon adenocarcinomas and in 10% of primary non-small cell lung carcinomas. In normal colon tissue, but not lung, there was evidence for age-related methylation of Chfr, suggesting that in some cases the tumor may have arisen from a methylated clonal precursor. Methylation was associated with loss of Chfr mRNA and protein expression in cancer cell lines. In cells with methylated Chfr, treatment with the demethylating agent 5-aza-2'-deoxycytidine resulted in re-expression of Chfr, and partial restoration of the prophase checkpoint. These results suggest that epigenetic inactivation of Chfr may be responsible for many of the checkpoint defects observed in human cancers.

Cardiac complications are not prominent features of snake bite and the clinical picture is usually dominated by neurological, haematological and vascular damage by the snake bite toxins. Myocardial involvement is seen on occasions and may rarely contribute to morbidity and mortality. T wave abnormalities are the most common manifestation of myocardial involvement, although ST segment depression, QRS prolongation and AV conduction defects may also be seen rarely. ECG changes are usually transient but when persistent they are attributed to direct myocardial damage due to the toxin [1], One such case who showed these rare ECG changes following snake bite is reported. Case Report 61 year old female patient, not a known case of ischaemic heart disease, hypertension, diabetes mellitus or any other illness was hospitalised with the history of snake bite over the left foot. She had tingling sensation and numbness over the limb up to knee joint associated with progressive swelling upto mid calf region in next 6 hours. Clinical examination revealed an elderly lady, afebrile, having irregularly irregular pulse of 1487min and BP110/70 mm Hg. Systemic examination was not contributory. Investigation showed abnormal coagulation parameters (BT-8′20” CT-25′), platelet count 1,80,000/cmm, Hbllgm/dl, TLC 7600/cmm, blood urea 30 mg/dl, serum creatinine 0.6 mg/dl, cardiac enzymes as available were SGOT 30 IU/1 and SGPT 18 IU/l. Serial estimation over the next 3 days did not show any abnormalities in enzyme levels. Other investigations like serum electrolytes, serum calcium, prothrombin time and X-ray chest were within normal limits. Fibrinogen and fibrinogen degradation product estimation could not be done. ECG showed an irregular rhythm, junctional ectopics. RBBB and ST depression of 1-4 mm (Fig-1). Patient was managed with anti-snake venom, xylocard and supportive measures. Pulse rate returned to normal in next 48 hours while coagulation parameters reverted to normalcy in 96 hours. Patient made an uneventful recovery. Serial ECG showed reversal to sinus rhythm in 48 hours. Open in a separate window Fig. 1 Cardiac involvement in snake bite

Recent analyses of global and gene-specific methylation patterns in cancer cells have suggested that cancers from different organs demonstrate distinct patterns of CpG island hypermethylation. Although certain CpG islands are frequently methylated in many different kinds of cancer, others are methylated only in specific tumor types. Because distinct patterns of CpG island hypermethylation can be seen in tumors from different organs, it seems likely that histological subtypes of cancer within a given organ may exhibit distinct methylation patterns as well. The goal of our study was to determine whether the patterns of CpG island hypermethylation could be used to distinguish between different histological subtypes of lung cancer. We analyzed the methylation status of 23 loci in 91 lung cancer cell lines using the quantitative real-time PCR method MethyLight. Genes PTGS2 (COX2), CALCA, MTHFR, ESR1, MGMT, MYOD1, and APC showed statistically significant differences in the level of CpG island methylation between small cell lung cancer (SCLC) and non-small cell lung cancer cell lines (NSCLC). Hierarchical clustering using a panel consisting of these seven loci yielded two major groups, one of which contained 78% of the SCLC lines. Within this group, a large cluster consisted almost exclusively of SCLC cell lines. Our results show that DNA methylation patterns differ between NSCLC and SCLC cell lines and suggest that these patterns could be developed into a powerful molecular marker to achieve accurate diagnosis of lung cancer.

We investigated the aberrant methylation profile of prostate cancers and correlated the data with clinical findings.Gene promoter methylation was analyzed in 101 prostate cancer samples. In addition, we analyzed 32 nonmalignant prostate tissue samples, which included 25 with benign disease, benign prostatic hypertrophy, or prostatitis, and 7 normal tissues adjacent to cancer. The methylation status of 10 genes was determined. The methylation index (MI) was calculated as a reflection of the methylated fraction of the genes examined.Methylation percentages of the genes tested in prostate cancers were: RARbeta, 53%; RASSF1A, 53%; GSTP1, 36%; CDH13, 31%; APC, 27%; CDH1, 27%; FHIT, 15%; p16(INK4A), 3%; DAPK, 1%; and MGMT, 0%. Methylation percentages in nonmalignant tissues were much lower. For clinicopathological correlations, we divided the cancer cases into low (6 or less) or high (7 or more) Gleason score (GS) groups, and into low (8 ng/ml or less) or high (greater than 8 ng/ml) preoperative serum prostate-specific antigen (PSA) groups. Methylation of RASSF1A, GSTP1, RARbeta, and CDH13 genes was significantly more frequent in the high GS group than in the low GS group. Methylation of RASSF1A, CDH1, and GSTP1 genes was significantly more frequent in the high PSA group than in the low PSA group. The median MIs were significantly higher in the high GS and the high PSA groups. According to the Spearman rank-correlation test, there was significant correlation between MI and GS (coefficient = 0.43, P0.0001) and the preoperative serum PSA (coefficient = 0.37, P = 0.0003).Our results indicate that the methylation profile of prostate cancers correlates with clinicopathological features of poor prognosis.