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Biomedical subjects

F M Scott

Publications and source records attributed to F M Scott.

At least 19 recordsLinked to original sources
PubMed2000

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.

Biomarkers, Tumor↗
PubMed2000

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.

Amidine-Lyases↗
PubMed2000

K-ras point mutation detection in lung cancer: comparison of two approaches to somatic mutation detection using ARMS allele-specific amplification.

BACKGROUND: The use of sensitive molecular techniques to detect rare cells in a population is of increasing interest to the molecular pathologist, but detection limits often are poorly defined in any given molecular assay. We combined the approaches of real-time quantitative PCR with ARMS(TM) allele-specific amplification in a novel assay for detecting mutant K-ras sequences in clinical samples. METHODS: ARMS reactions were used to detect seven commonly occurring mutations in the K-ras oncogene. These mutations produce amino acid changes in codon 12 (Gly to Ala, Arg, Asp, Cys, Ser, or Val) and codon 13 (Gly to Asp). A control reaction was used to measure the total amount of amplifiable K-ras sequence in a sample so that the ratio of mutant to wild-type sequence could be measured. Quantitative data were confirmed for a selection of samples by an independent cloning and sequencing method. The assay was used to analyze 82 lung tumor DNA samples. RESULTS: The assay detected K-ras mutations in 44% of adenocarcinomas, which is equivalent to frequencies reported in the literature using ultrasensitive techniques. Forty-six percent of squamous carcinomas were also positive. The ratio of mutant sequence in the tumor DNA samples was 0.04-100%. CONCLUSIONS: The assay is homogeneous, with addition of tumor DNA sample being the only step before results are generated. The quantitative nature of the assay can potentially be used to define the analytical sensitivity necessary for any specified diagnostic application of K-ras (or other) point mutation detection.

Alleles↗
PubMed1999

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.

Adenocarcinoma↗
PubMed1998

Correlation of expression of bombesin-like peptides and receptors with growth inhibition by an anti-bombesin antibody in small-cell lung cancer cell lines.

The murine anti-bombesin monoclonal antibody, 2A11, has been demonstrated to inhibit growth of some small-cell lung cancer (SCLC) cells in nude mice xenografts and in a clinical trial. To determine if the expression of bombesin-like peptides (BLP) and their receptors (GRP-R and NMB-R) correlate with an in vitro response to 2A11, we measured these parameters in seven SCLC cell lines. Gastrin releasing peptide (GRP) mRNA was detected in three of seven cell lines (NCI-H69, NCI-H345, NCI-H510) and neuromedin B (NMB) mRNA was detected in all seven lines using an RNase protection assay (RPA). Immunoreactive BLP was detected in the cell pellets of all lines (range 0.11-59.90 pmol/mg protein) by a solid phase GRP radioimmunoassay (RIA) using 125I-labeled 2A11. RPA detected GRP-receptor mRNA in two cell lines (NCI-H69 and NCI-H345) and NMB-receptor in three lines (NCI-H345, NCI-H510, and NCI-H660). Reverse transcriptase-PCR confirmed the presence of receptor mRNA in these lines and detected NMB-receptor in an additional three lines (NCI-H69, NCI-H82, and NCI-H187). Calcium mobilization in response to BLP stimulation was detected in the six cell lines expressing either GRP-R or NMB-R mRNA but not in NCI-N417, which had no detectable BLP-receptor. 2A11 (5 microg/ml) inhibited colony formation by 26-61% after 2 weeks in all cell lines except NCI-N417. Thus, growth inhibition by 2A11 requires the presence of at least one BLP-receptor. These findings may be useful in selecting patients with SCLC for treatment with 2A11.

Animals↗
PubMed1997

High frequency of K-ras codon 12 mutations in bronchoalveolar lavage fluid of patients at high risk for second primary lung cancer.

A high frequency of K-ras mutations may indicate preneoplastic changes in the bronchial epithelium as a result of genotoxic injury. With the use of sensitive detection techniques, we report a higher prevalence of K-ras mutations in bronchoalveolar lavage than has been reported previously for lung cancer. A PCR/ligase chain reaction technique was used to determine K-ras codon 12 mutations in a group of 52 bronchoalveolar lavage specimens from patients at risk of a second lung cancer. Of the specimens examined, 84% contained at least one mutation in K-ras codon 12, corroborated by an allele-specific hybridization method. These results suggest that point mutations in K-ras codon 12 are widespread in the bronchial epithelium. Based on these preliminary findings, further evaluation of this efficient sensitive assay to monitor K-ras status should be conducted in larger clinical cohorts where clinical outcomes will ultimately be available. Such a trial will define the utility of K-ras codon 12 mutation status as a marker of lung cancer.

Bronchoalveolar Lavage Fluid↗
PubMed1996

Purification and characterization of a protein that permits early detection of lung cancer. Identification of heterogeneous nuclear ribonucleoprotein-A2/B1 as the antigen for monoclonal antibody 703D4.

We have reported that a mouse monoclonal antibody 703D4, detects lung cancer 2 years earlier than routine chest x-ray or cytomorphology. We purified the 703D4 antigen to elucidate its role in early lung cancer biology, using Western blot detection after SDS-polyacrylamide gel electrophoresis. Purification steps included anion exchange chromatography, preparative isoelectric focusing, polymer-based C18-like, and analytical C4 reverse phase high performance liquid chromatography. After 25-50,000-fold purification, the principal immunostaining protein was > 95% pure by Coomassie staining. The NH2 terminus was blocked, so CNBr digestion was used to generate internal peptides. Three sequences, including one across a site of alternate exon splicing, all identified a single protein, heterogeneous nuclear ribonucleoprotein-A2 (hnRNP-A2). A minor co-purifying immunoreactive protein resolved at the final C4 high performance liquid chromatography step is the splice variant hnRNP-B1. Northern analysis of RNA from primary normal bronchial epithelial cells demonstrated a low level of hnRNP-A2/B1 expression, consistent with immunohistochemical staining of clinical samples, and increased hnRNP-A2/B1 expression was found in lung cancer cells. hnRNP-A2/B1 expression is under proliferation-dependent control in normal bronchial epithelial cell primary cultures, but not in SV40-transformed bronchial epithelial cells or tumor cell lines. With our clinical data, this information suggests that hnRNP-A2/B1 is an early marker of lung epithelial transformation and carcinogenesis.

Amino Acid Sequence↗
PubMed1996

Expression in human lung cancer cell lines of genes of prohormone processing and the neuroendocrine phenotype.

Lung tumor cells and cell lines, principally the histologically classified small cell lung cancer, are characterized by the expression of neuroendocrine (NE) features including AADC (aromatic amino acid decarboxylase, previously called DOPA decarboxylase) and the production of many peptide hormones. The general mechanisms by which most aspects of the NE phenotype affect the clinical behavior of lung tumor cells are unknown, but it is well recognized that peptide hormones can have systemic effects (paraneoplastic syndromes) and several have been shown to be autocrine growth factors for cancer cells. In order to determine the relationship between expression of different aspects of the NE phenotype in lung cancer cell lines, we have compared expression of a gene required for biosynthesis of some active peptide hormones (PAM, peptidylglycine alpha-amidating monooxygenase) to the gene for AADC in 32 lung cancer cell lines. Expression of these genes was quantified by both steady state Northern blot analysis and radiochemical enzymatic activity measurements. To ensure a range of expression of NE markers, non-small cell lung cancer (NSCLC) cell lines were chosen to include several which had previously been shown to express NE markers, and several small cell lung cancer (SCLC) cell lines with previous low levels of AADC were included. PAM enzyme activity and Northern blot analysis showed a two to three log variation in levels of expression in both the small cell and non-small cell lines. A smaller range was found for AADC expression. Using the highly sensitive PAM enzyme assays, all cell lines were found to express detectable PAM. PAM activities were secreted into the growth medium of all cell lines. There was no simple correlation apparent between AADC and PAM gene expression in the lung cancer cell lines. However, classic small cell lines demonstrated high levels of expression of both PAM and AADC genes, as did the carcinoid subset of the NSCLC lines. NSCLC lines expressed levels of PAM mRNA and enzyme activities equivalent to those of SCLC but had infrequent expression of AADC (principally only carcinoid NSCLC expressed AADC). These data demonstrate that separate aspects of the NE phenotype can be differentially expressed in lung cancer histological sub-types. Expression of PAM enzymes in all sub-types of lung cancer suggests that peptide prohormone activation may be a common mechanism for autocrine growth stimulation even in non-Ne NSCLC cell lines, or may reflect maintenance in cell lines of a common pathway of lung tumor promotion.

Biomarkers, Tumor↗
PubMed1996

Peptide amidating activity in human bronchoalveolar lavage fluid.

Monitoring respiratory epithelial biology may reveal individuals with incipient lung cancer. The expression of neuroendocrine (NE) markers in pulmonary epithelium is thought to be central to lung development, repair of injury and may contribute to carcinogenesis. In this study, we evaluate several candidate NE markers to determine the feasibility of prospective analysis of clinical specimens. The potential NE markers include the enzyme L-DOPA decarboxylase (DDC), the neuropeptide gastrin releasing peptide (GRP), and peptidyl-glycine alpha-amidating monooxygenase (PAM), the bifunctional enzyme responsible for the final bioactivation step of many neuropeptides. A comparison of PAM activity and DDC levels in 30 lung cancer cell lines indicated that peptide amidating activity may be an indicator of NE status. Bronchoalveolar lavage (BAL) fluid from subjects at risk of developing second primary lung cancer and from volunteers was obtained. The activity of the first PAM enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), ranged from not detectable to 507 pmol/h/mg protein in 57 specimens. The second PAM enzyme, peptidylamidoglycolate lyase (PAL), ranged from not detectable to 414 pmol/h/mg protein in 56 specimens. Using cluster analysis by the average linkage method, a group of enzyme values with PHM greater than 230 pmol/h/mg protein was determined. Long-term follow-up of these patients for new second primary lung cancers may help to determine the potential predictive value of PAM detected in the BAL fluid.

Biomarkers, Tumor↗
PubMed1993

Biochemical characterization of peptide alpha-amidation enzyme activities of human neuroendocrine lung cancer cell lines.

Peptide alpha-amidation is a posttranslational modification of approximately half of all endocrine and neuroendocrine peptide hormones, including several hormones with mitogenic effects for tumor cells, and is typically essential for complete hormonal bioactivity. alpha-Amidated peptide hormones have been reported to be autocrine growth factors for small cell lung cancer cells. We report here that a variety of human lung tumor cell lines express both enzymes required for the two-step conversion of inactive glycine-extended peptides into their active COOH-terminal alpha-amide analogues. Human tumor cell peptidylglycine alpha-amidation enzymes are present in multiple molecular forms. Both proteins are metalloenzymes which are present at highest concentrations in secretory granules in neuroendocrine cell lines. The expression of these enzymes is positively correlated with expression of other markers of the neuroendocrine phenotype, such as DOPA decarboxylase. Peptidylglycine alpha-amidating enzyme-specific activities are approximately 50-fold higher in extracts of endocrine cell lines (lung small cell and carcinoid) than of nonendocrine lines. Biochemical characterization of the peptidylglycine alpha-amidating enzymes will enable development of tools for detection of endocrine processes in the early stages of neoplasia and for interruption of autocrine stimulation pathways in tumor cells.

Amidine-Lyases↗
PubMed1992

Candidate biomarkers for application as intermediate end points of lung carcinogenesis.

The need for validated intermediate end point markers to facilitate lung cancer chemointervention research is compelling. Three major classes of lung markers are relevant for this application. Since lung cancer includes four distinct histologies, markers that map degrees of histologic differentiation are important. Many of the markers for squamous differentiation overlap with the candidates for application in the study of head and neck cancer. Production of tissue-specific cell products especially for surfactant or CEA is of interest, because the gene structure is known and many differentiation-related polymorphisms exist. This strategy would be useful for adenomatous type tissue. A second type of marker is the broad group of differentiation markers. The carbohydrate or blood group-like antigens comprise a representative example. Carbohydrate structures are expressed in a specific sequence during fetal processes, and this sequence appears to reverse with the development of a cancer. Retrodifferentiation of specific differentiation markers is the basis of a major effort to effect earlier lung cancer detection using sputum immunocytochemistry. The final class includes markers which affect either positive or negative aspects of growth. Candidates in this area include growth factors or their receptors, or genes that regulate growth. If the intermediate end point marker reflects tumor biology and that biology is in the causal path of tumor progression, serial observation of that parameter should indicate the success of the intervention. In all three of these examples, the clinical material to be analyzed could be sputum specimens, bronchial biopsies or resected lung tissue. Systematic analysis of these markers in context of intervention trials is required to validate their utility.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoma↗
PubMed1991

Homotypic and heterotypic serological responses to rotavirus neutralization epitopes in immunologically naive and experienced animals.

Gnotobiotic or specific-pathogen-free animals with no previous exposure to rotavirus were vaccinated with strain UK, serotype G6. The highest serological response was to homologous virus; significant but lower responses occurred to viruses with either VP4 or VP7 related to that of vaccine virus; responses to other viruses were of low titer or infrequent. Adult cows vaccinated with UK virus produced increased titers of antibody to all rotavirus serotypes. The increases in titer to homologous virus and to other natural and reassortant viruses sharing VP7 with the vaccine virus were significantly higher than those to all other viruses. These results suggest the presence of common epitopes which are not well recognized in primary infections.

Animals↗
PubMed1991

Alpha-amidation of peptide hormones in lung cancer.

Small-cell lung cancer (SCLC), the most common neuroendocrine tumor in humans, provides an excellent model system for analyzing the role of growth factors in lung cancer. SCLCs secrete a wide range of peptide hormones, including some that stimulate tumor cell growth, such as gastrin-releasing peptide and insulin-like growth factor I. Many of these peptides are synthesized as prohormones that acquire biological activity only after specific post-translational modifications. Here, we review our current understanding of the biological role of neuroendocrine peptides in lung carcinogenesis and consider how a mechanistic knowledge of one particular modification, carboxy-terminal alpha-amidation, may permit identification of novel growth factors for lung cancer cells. We also describe potential applications of this knowledge as a basis for prevention-oriented approaches to the disease.

Amides↗
PubMed1991

Lung cancer: rational strategies for early detection and intervention.

The current mortality rate for lung cancer, which approaches 90% at two years, is not decreasing despite intensive clinical trials attempting to improve systemic therapy. New drug discoveries and dose intensification approaches have not resulted in more effective anti-tumor control. An alternative approach using sputum immunocytology for early lung cancer detection was recently reported. The proposed basis for this early detection reflects the underlying biology of immunodominant carbohydrate tumor-associated antigens, which are recognized as a class of oncofetal antigens. The process of bronchial epithelial carcinogenesis is associated with an evolution of carbohydrate display which reverses the sequence originally associated with fetal organ development. By elucidating this pattern of fetal carbohydrate display, a precise map of stage of bronchial carcinogenesis may emerge.

Antigens, Neoplasm↗
PubMed1990

A comparison of bovine coronavirus strains using monoclonal antibodies.

Eight monoclonal antibodies (MAbs) were raised against a bovine coronavirus (B.C.V.) which had been isolated in Scotland and was designated S2. The MAbs were divided into two groups on the basis of their reactions with S2 virus in indirect immunofluorescence (I.F.), neutralisation and haemagglutination inhibition (H.A.I.) tests. Five of the MAbs were positive by all three tests but failed to bind to proteins in Western immunoblotting experiments. The remaining three MAbs were positive in I.F. tests only, two of which were shown to bind to the 52K nucleocapsid protein by Western immunoblotting. Different patterns of antigen distribution within infected cells were demonstrated when the MAbs were used in the I.F. test. However only minor strain variations were detected by I.F. and H.A.I. tests when the MAbs were tested against each of five cell culture adapted strains of B.C.V. Twenty-nine isolates of B.C.V. have been grown in neonatal calf tracheal organ cultures: attempts are being made to further characterize these isolates.

Animals↗
PubMed1990

Pneumonia of lambs following inoculation of isolates of sheep herpesvirus (caprine herpesvirus 1) of different DNA genotypes.

One-day-old, specific pathogen-free lambs, were equally susceptible to infection with three isolates of caprine herpesvirus 1 (CHV1). One of these isolates was genomically different by DNA analysis. Lesions, which were confined to the lung, ranged from a mild interstitial reaction to widespread consolidation. CHV1 was recovered from lungs and less commonly from liver and adrenal gland. Three-week-old SPF lambs were also successfully infected with CHV1. Histopathological findings were similar to those in 1-week-old lambs.

Animals↗