Biomedical subjects

D G Beer

Publications and source records attributed to D G Beer.

At least 37 records · Page 2Linked to original sources

PubMed1998

Abundant expression of the intestinal protein villin in Barrett's metaplasia and esophageal adenocarcinomas.

Villin is a cytoskeletal protein that is involved in the formation of brush-border microvilli in normal small intestine and colon epithelium. This protein is present in Barrett's metaplasia but is reported not to be expressed in Barrett's adenocarcinoma. In this study, we analyzed villin protein expression in Barrett's metaplasia and in both Barrett's adenocarcinomas and tumors of the gastric cardia. Immunohistochemical analysis was used to evaluate the expression and cellular localization of the villin protein in 21 cases of Barrett's metaplasia, 30 cases of Barrett's adenocarcinoma, 16 cases of gastric cardia adenocarcinoma, and eight cases of adenocarcinoma of the distal esophagus. Southern, northern, and western blot analyses were used to evaluate the potential mechanisms for regulation of villin protein expression. Villin protein expression was observed in 21 of 21 cases (100%) of intestinal-type Barrett's metaplasia and in 28 of 30 cases (93%) of Barrett's adenocarcinoma and was thus highly expressed in these tumors. Northern blot analysis demonstrated villin mRNA (3.5 and 2.7 kb) in both villin-positive Barrett's metaplasia and adenocarcinomas. Western blot analysis with the antibody used for immunohistochemical analysis confirmed the presence of a single villin protein band of 95 kDa. Abundant villin expression also was present in both adenocarcinoma of the gastric cardia (13 of 16 cases; 81%) and distal esophageal adenocarcinomas of unknown origin (six of eight cases; 75%). The intestinal brushborder enzyme sucrase isomaltase was found to be present in only 22 of 46 cases (48%) of the adenocarcinomas that expressed villin. We concluded that the protein villin is highly expressed in Barrett's adenocarcinomas and is well maintained in these and other esophageal tumors.

Adenocarcinoma↗

PubMed1998

Unique expression patterns and alterations in the intestinal protein villin in primary and metastatic pulmonary adenocarcinomas.

The identification of markers that distinguish primary pulmonary adenocarcinomas from pulmonary adenocarcinomas secondary to the digestive tract would be clinically important. Villin, a specific marker in digestive-tract malignancies, was evaluated in 57 pulmonary adenocarcinomas, six samples of proximal bronchial tissue, and five metastatic pulmonary adenocarcinomas (three colon and two esophageal adenocarcinomas) by using immunohistochemical and molecular analyses. Villin was expressed in 31.6% (18 of 57) of the pulmonary adenocarcinomas and showed either a diffuse cytoplasmic pattern (10.5%) or a primary cytoplasmic pattern with minor brush-border staining (21.1%). However, none of those samples demonstrated the primary brush-border staining pattern that was characteristic of all five of the metastatic digestive-tract adenocarcinomas. There was a significant difference in the positive brush-border staining pattern between the primary and metastatic pulmonary adenocarcinomas (P < 0.002). Villin protein was expressed in bronchial epithelial cells, and villin mRNA was detected by reverse transcription-polymerase chain reaction. Northern analysis demonstrated 3.5- and 2.7-kb villin mRNAs in villin protein-positive tumors, but villin mRNA was not detected in non-tumorous lung tissue, indicating the transcriptional upregulation of villin in lung tumors. An additional smaller-sized mRNA (1.8 kb) was observed in six of 10 pulmonary adenocarcinomas and in the bronchoalveolar carcinoma cell line A549. Two small villin mRNAs were cloned from the cell line A549 and were found to represent an alternatively spliced (exon 8-exon 14) 1.85-kb mRNA and a 1.8-kb mRNA that was missing a portion of the 5' region (exon 1-exon 9) of the native villin mRNA. These studies demonstrated that the pattern of villin expression and the presence of altered villin mRNAs may be useful markers for pulmonary adenocarcinomas as well as provide support for the potential origin of villin-expressing tumors from bronchial epithelial cells.

Adenocarcinoma↗

PubMed1997

Fas/APO-1 (CD95) is not translocated to the cell membrane in esophageal adenocarcinoma.

This study describes Fas (CD95) expression in Barrett's esophagus, adenocarcinomas of the esophagus, and three esophageal adenocarcinoma cell lines. Immunohistochemical analysis of Barrett's esophagus demonstrated cell surface expression of Fas protein. In contrast, 30.5% of esophageal adenocarcinomas examined by immunohistochemical analysis demonstrated faint cytoplasmic staining, and 69.5% were negative for Fas. Similar levels of Fas mRNA were identified in tumors compared to mRNA levels in esophageal squamous mucosa or Barrett's esophagus. An approximately Mr 48,000 Fas protein was identified by Western blot analysis in tumors that were negative for Fas expression by immunohistochemical analysis. The esophageal adenocarcinoma cell line Seg-1 was negative for Fas expression by immunohistochemical analysis, but Western blot analysis demonstrated abundant, appropriately sized Fas protein. In agreement with the immunohistochemical analysis, flow cytometry of Seg-1 showed minimal amounts of Fas on the cell surface, which correlated with resistance to Fas-mediated apoptosis. No mutations in the Seg-1 Fas coding sequence or exon 1 were identified by sequence analysis. This was confirmed by transient transfection of COS cells with expression vectors generated from the Seg-1 Fas cDNA, which resulted in cell surface expression of the Fas protein. Stable transfection of Seg-1 with a Fas expression vector did not result in efficient Fas expression on the cell surface. Seg-1 cells, transiently transfected with a Fas-FLAG expression vector and examined for protein expression using confocal microscopy and an anti-FLAG antibody, showed that the Fas-FLAG protein was not present on the cell surface but was present in the cytoplasm. Taken together, these results indicate that expression of Fas on the cell surface by esophageal adenocarcinoma is reduced. In an esophageal adenocarcinoma cell line, wild-type Fas protein is retained in the cytoplasm, and this correlates with resistance to Fas-mediated apoptosis. The retention of wild-type Fas protein within the cytoplasm may represent a mechanism by which malignant cells evade Fas-mediated apoptosis.

Adenocarcinoma↗

PubMed1997

Frequent deletions of FHIT and FRA3B in Barrett's metaplasia and esophageal adenocarcinomas.

The FHIT gene, which spans the common fragile site FRA3B, has been shown to produce aberrant transcripts in a variety of tumor types. Homozygous deletions within the FHIT locus have been detected only in tumor-derived cell lines and LOH has been described in numerous primary tumors. Based on these findings and its location on 3p, FHIT has been proposed as a tumor suppressor gene. To further study the relationship of FRA3B to the findings regarding the FHIT gene and to determine the extent of FHIT mRNA alterations in early stages of tumor development, the status of the FHIT gene was evaluated in the premalignant condition of Barrett's esophagus and associated esophageal adenocarcinomas. FHIT expression was investigated by RT-PCR in normal esophageal, Barrett's metaplasia and adenocarcinoma tissues from 15 patients. Alterations of FHIT transcripts were observed in 12/14 (86%) of Barrett's metaplasia and in 14/15 (93%) of the adenocarcinomas from the same patients. Characterization of the altered transcripts revealed FHIT mRNA lacking one or more exons, with deletion of exons 5-7 being most frequent. Analysis of genomic DNA from 20 patients showed homozygous deletions involving exon 5 of FHIT in 4/20 (20%) esophageal adenocarcinomas, and 7/20 (35%) tumors demonstrated hemizygous loss. Genomic deletions also involved the BE758-6 locus, indicating that a large region is deleted. Fluorescence in situ hybridization (FISH) analysis demonstrated that this region of deletion is localized within FRA3B. Our results extend the range of tumor types in which altered FHIT transcripts have been demonstrated and show that these alterations can be seen in the premalignant stage of esophageal tumor development. These results indicate that the fragility and recombination-prone nature of FRA3B is related to tumor-specific chromosomal instability affecting the FHIT gene in esophageal adenocarcinoma development.

Acid Anhydride Hydrolases↗

PubMed1997

Detection of p53 nuclear protein accumulation in brushings and biopsies of Barrett's esophagus.

Alterations in the tumor suppressor gene p53 may represent a useful prognostic marker of premalignant or malignant disease in Barrett's dysplasia or esophageal adenocarcinoma. Immunohistochemistry was used to establish the ability to detect nuclear accumulation of altered p53 protein in esophageal brushings as well as biopsies, and to examine for p53 alterations in a group of 18 patients with Barrett's esophagus enrolled in a surveillance and endoscopy program, p53 protein accumulation was easily detected in esophageal brushings, and the results correlated well with matched biopsies (9/11). In patients enrolled in surveillance endoscopy, 1 brushing of 22 was positive for p53 protein accumulation. In this patient, who received preoperative radiation and chemotherapy, the positive p53 result correlated with positive cytology for residual adenocarcinoma. All Barrett's esophagus brushings negative for p53 protein were benign by cytologic, morphologic criteria. The immunohistochemical detection of p53 alterations in esophageal brushing and biopsy specimens may provide useful information in patients undergoing surveillance for esophageal dysplasia and adenocarcinoma.

Adenocarcinoma↗

PubMed1996

Loss of glucocorticoid-dependent growth inhibition in transformed mouse lung cells.

Transformed A5 mouse lung cells were examined for mechanisms that may explain their loss of glucocorticoid-induced growth inhibition. These cells were compared to nontransformed C10 mouse lung cells, which retain this response. Southern blot analysis revealed no major differences in the amount or pattern of restriction fragments for the glucocorticoid receptor (GR) gene between the responsive and nonresponsive cells. Northern blot analysis demonstrated that both cell lines expressed GR mRNA at similar levels and that these mRNAs had similar relative stabilities. The mRNA from both cell lines was used for reverse transcription-polymerase chain reaction amplification and direct sequencing with primers for different regions of the GR cDNA. A conservative mutation previously shown not to affect receptor function was detected within the DNA-binding domain region of the GR from both cell lines. Because of the ability of the transcription factors for activator protein-1 to antagonize GR function, c-jun and c-fos mRNA levels were examined. A5 cells were found to have higher levels of c-jun mRNA than C10 cells both during active cell growth and after serum starvation. Stable transfection of the nonresponsive A5 cells with a rat GR expression vector (A5GR7) resulted in strong glucocorticoid-induced growth inhibition, demonstrating that these cells retain the ability to be growth inhibited by these steroids. The A5GR7 transfectants also had higher mouse mammary tumor virus (MMTV)-chloramphenicol acetyltransferase (CAT) activity than the parental A5 cells and lower levels of c-jun during active cell growth. Transient transfection of the C10 cells with c-jun expression vector strongly reduced glucocorticoid-inducible MMTV-CAT activity. These results suggest that the transformed A5 cells apparently contain functional GR but that the high level of c-jun mRNA expression (probably resulting from the activated Ki-ras allele in these cells) may antagonize their ability to respond to the growth-inhibitory signaling of glucocorticoids.

Animals↗

PubMed1996

Influence of the glucocorticoid receptor on c-fos inducibility in activated ras-containing mouse lung cells.

Glucocorticoids inhibit the growth and promote the differentiation of normal lung cells. Transformed A5 mouse lung cells containing an activated Ki-ras gene are not responsive to glucocorticoid-induced growth inhibition and demonstrate increased cell proliferation. Activated ras genes may lead to constitutive activation of genes, such as the activating protein 1 (AP-1) transcription factor components fos and jun, which are downstream in the ras signal-transduction pathway. A5 cells and A5GR, a stable A5 transfectant containing excess copies of the glucocorticoid receptor (GR) gene, were examined for potential alterations in AP-1 that accompany the restoration of glucocorticoid-dependent growth inhibition. The established ability of the GR to antagonize AP-1 activity led us to examine the regulation and inducibility of c-fos and c-jun in these cells. Nontransformed C10 lung cells were found to have higher and more inducible AP-1 activity than the transformed A5 cells. The level of AP-1 activity could be reduced in C10 cells by transient transfection of constitutive fos and jun expression vectors. In A5 cells, stimulation with factors that activate the serum-response element on the fos promoter and induce c-fos mRNA had little effect on AP-1 activity, whereas treatment with 12-O-tetradecanoylphorbol-13-acetate, which acts at the fos-AP-1 binding sequence site on the fos promoter, efficiently induced c-fos mRNA. The c-fos mRNA in A5GR cells, however, was not inducible with all treatments, suggesting that one potential mechanism by which the GR restores glucocorticoid-induced growth inhibition in these cells may involve the desensitization of additional 12-O-tetradecanoylphorbol-13-acetate-inducible elements of the fos promoter.

Animals↗

PubMed1996

Detection of Barrett's adenocarcinoma of the gastric cardia with sucrase isomaltase and p53.

BACKGROUND: Routine surveillance for dysplastic epithelium in patients with Barrett's esophagus has markedly improved prognosis. Many patients with short segments of Barrett's mucosa near the esophagogastric junction remain undiagnosed and at risk for the development of Barrett's adenocarcinomas (BA). Sucrase isomaltase (SI), an intestinal enzyme, is highly expressed in intestinal-type Barrett's mucosa and frequently expressed in dysplastic Barrett's mucosa and BA. Sucrose isomaltase is not expressed in normal esophageal or gastric mucosa. Alterations in the p53 tumor suppressor gene are frequent events in dysplastic Barrett's mucosa and BA and result in nuclear protein accumulation. The purpose of this study was to determine the presence or absence of these markers of Barrett's mucosa in adenocarcinoma of the esophagogastric junction or cardia. METHODS: Expression of SI and p53 were examined in 40 BAs and 25 cardia adenocarcinomas using immunohistochemical techniques. RESULTS: Sucrose isomaltase analysis revealed positive staining in 55% (22/40) of the BAs and 44% (11/25) of the cardia adenocarcinomas. Of 14 cardia adenocarcinomas that were SI negative, 100% (14/14) had no associated Barrett's mucosa. However, in 21 cardia adenocarcinomas with no associated Barrett's mucosa, 7/21 (33%) were SI positive. This suggests that SI-positive tumors may represent BA without the standard definition of Barrett's esophagus being met. P53 was present in 65% of BAs and 64% of cardia adenocarcinomas, demonstrating the importance and similarity of this gene alteration in both tumor types. Staining was positive for SI or p53 in 77% (50/65) of all tumors. Tumors of lower stage expressed SI more often than higher stage tumors. CONCLUSIONS: These data suggest that a subset of cardia adenocarcinomas represent BAs. Surveillance endoscopy incorporating additional esophagogastric junction biopsies and assessment of SI or p53 may improve detection of intestinalized Barrett's mucosa and early dysplastic changes.

Adenocarcinoma↗

PubMed1995

Effect of increased glucocorticoid responsiveness in transformed mouse lung cells.

Many transformed mouse lung cells, including LM2 cells, contain activating mutations in the Ki-ras gene and show reduced responsiveness to growth inhibition by glucocorticoids. LM2GR cells, which are LM2 cells stably transfected with a rat glucocorticoid receptor (GR) gene, were used to determine whether increasing glucocorticoid responsiveness can influence aspects of the transformed phenotype. LM2GR cells grew slower and had a lower final saturation density than the parental LM2 cells. Expression of growth-related genes was examined by northern blot analysis. The cells were serum-deprived and treated with fetal bovine serum (FBS), steroid-stripped FBS (ssFBS), dexamethasone, or 12-O-tetradecanoylphorbol-13-acetate. The level and pattern of Ki-ras mRNA expression was similar in both LM2 and LM2GR cells, but histone H4 mRNA was expressed in a more regulated fashion in LM2GR cells. The induction of c-jun and c-fos mRNA expression lasted longer in the LM2GR cells treated with ssFBS; however, the maximal induction was greater in the LM2 cells treated with FBS. LM2GR cells demonstrated similar activator protein-1 (AP-1) activity but higher GR activity than LM2 cells as determined by using AP-1-chloramphenicol acetyltransferase (CAT) and mouse mammary tumor virus-CAT transient transfection assays, consistent with the higher level of GR mRNA in LM2GR cells. Both cell lines exhibited the ability to grow in soft agar and to form tumors in nude mice. These results indicate that introduction of a functional GR transgene into LM2 cells can increase glucocorticoid responsiveness and alter the expression of genes involved in growth regulation but cannot overcome anchorage-independent cell growth or tumorigenicity, apparently because of the presence of an activated Ki-ras gene.

Animals↗

PubMed1995

Expression and activation of erbB-2 and epidermal growth factor receptor in lung adenocarcinomas.

ErbB-2 and EGFR (epidermal growth factor receptor) are expressed in lung adenocarcinomas and associated with a poor prognosis. Immunocytochemical analysis revealed erbB-2 and EGFR coexperession as a characteristic feature of most lung adenocarcinomas, and at levels of receptor expression present in bronchial epithelial cells. In primary lung tumours and cell lines, erbB-2 detected using Western blot analysis demonstrated low-level phosphotyrosine staining of the 185 kDa band, as compared with breast cancer cell lines. A549 and A427 lung adenocarcinoma cells treated with neu differentiation factor (NDF) showed increased erbB-2 phosphotyrosine staining, but to a much lesser extent than breast cancer cells. The lung cells were examined for expression of the potential autocrine growth factors NDF and transforming growth factor alpha (TGF-alpha) by Northern blot analysis. Both NDF and TFG-alpha mRNA were abundantly expressed in the A549 cells. NDF mRNA was highest during active cell proliferation and decreased in confluent cells or after treatment with the growth-inhibitory steroid dexamethasone. Primary tumours and cell lines expressed EGFR, showing higher basal level phosphotyrosine staining than erbB-2. Treatment with NDF and EGF (epidermal growth factor) stimulated cell growth, and in A549 cells the presence of both factors provided an additive increase in cell growth. The growth stimulus that ligand-activated erbB-2 and EGFR provides to lung adenocarcinoma cells may establish a background of continued cell proliferation over which other critical transforming events may occur.

Adenocarcinoma↗

PubMed1995

E-cadherin expression in primary and metastatic thoracic neoplasms and in Barrett's oesophagus.

Reduced expression of E-cadherin, a Ca(2+)-dependent cell adhesion molecule present in normal epithelium, has been associated with invasive and metastatic cancer. Immunohistochemistry was used in examining the relationship between E-cadherin expression and stage in 59 oesophageal and 52 lung cancers. Advanced-stage oesophageal cancers were associated with both reduced and disorganised E-cadherin expression (P < 0.01). Advanced-stage lung adenocarcinomas generally exhibited disorganised or reduced E-cadherin expression, but no statistical association between expression pattern and stage was found (P > 0.05). No differences in stage were seen between tumours with reduced or disorganised E-cadherin expression. Altered E-cadherin expression was detected in dysplastic, non-invasive Barrett's oesophagus. Importantly, high-level E-cadherin expression was detected in 17 of 17 lymph nodes containing metastatic cancer. E-cadherin mRNA expression was decreased in tumours with reduced protein expression, but not in tumours with disorganised expression. Expression of alpha-catenin mRNA, an E-cadherin-associated protein, was detected in tissues with altered E-cadherin protein expression. Reduced and disorganised expression of E-cadherin appear to be related to transcriptional and post-translational events respectively, and both appear to represent altered cell adhesion associated with invasion and metastasis in thoracic neoplasms.

Adenocarcinoma↗

PubMed1994

Intestinal differentiation and p53 gene alterations in Barrett's esophagus and esophageal adenocarcinoma.

The development of esophageal adenocarcinoma is frequently associated with intestinal-type Barrett's metaplasia. Barrett's metaplasia and esophageal adenocarcinomas were examined for expression of the intestinal brush-border-associated hydrolase aminopeptidase N (APN). APN mRNA was detected by utilizing the reverse transcription polymerase chain reaction (RT-PCR) in 50% of Barrett's metaplasia specimens and in 26% of esophageal adenocarcinomas. APN protein was detected by utilizing immunohistochemistry in 84% of Barrett's metaplasia specimens and in 71% of adenocarcinomas, although a decrease or loss of APN protein was sometimes observed in dysplastic Barrett's metaplasia and adenocarcinomas. Alterations in the p53 tumor-suppressor gene have previously been found in both dysplastic Barrett's mucosa and esophageal adenocarcinomas. The same specimens analyzed for APN were examined for the nuclear accumulation of the p53 protein. Utilizing immunohistochemistry, p53 staining was detected in 42% of Barrett's metaplasia specimens, most of which were dysplastic, and in 58% of adenocarcinomas. In the samples positive for p53 protein, gene mutations in exons 5, 7 and 8 were detected by utilizing single-strand conformation polymorphism analysis (SSCP) in 1 Barrett's metaplasia specimen and 6 adenocarcinomas. In Barrett's metaplasia, there was an inverse correlation between APN protein expression and p53 protein accumulation (p < 0.05) suggesting a link between genetic alterations and loss of this marker. The analysis of markers of intestinal differentiation with markers of disease progression may prove to be a useful approach for studying carcinogenesis in Barrett's metaplasia.

Adenocarcinoma↗

PubMed1994

Inhibition of tumor necrosis factor production by lymphocytes from anti-TNF antibody-treated, cardiac-allografted rats.

Tumor necrosis factor-alpha (TNF) is a multifunctional cytokine involved in the immunopathologic consequences of allograft rejection. We have previously demonstrated that anti-TNF antibody treatment prolongs cardiac allograft survival in rats. To elucidate the mechanism of anti-TNF antibody in modulating the immune response, we investigated TNF production by spleen and lymph node cells from anti-TNF antibody-treated Lewis rats who received MHC-mismatched Brown Norway rat cardiac allografts. In 10 untreated rats, cardiac allografts were rejected at 6.8 +/- 0.6 days after transplantation (mean +/- SD). Anti-TNF antibody treatment enhanced graft survival to 12.7 +/- 1.4 days (P < 0.001 vs controls). In other anti-TNF antibody-treated recipients spleen and lymph node cells were isolated on Day 5 after transplant. TNF production was measured and showed significantly less TNF than those from untreated (no anti-TNF antibody), transplanted recipient rats (28.7 u/10(6) spleen cells vs 76.4 u/10(6) spleen cells at 2 hr and 4.6 u/10(6) lymph node cells vs 9.2 u/10(6) lymph node cells at 24 hr). Furthermore, following lipopolysaccharide stimulation, spleen cells from anti-TNF-treated rats again produced significantly less TNF than those from untreated transplanted rats (68.9 u/10(6) cells vs 189.4 u/10(6) cells at 2 hr). Finally, with allogeneic cell stimulation, anti-TNF treated rats again produced significantly less TNF than untreated transplanted rats (spleen cells, 2.2 u/10(6) cells vs 40.4 u/10(6) cells at 24 hr; lymph node cells, 1.2 u/10(6) cells vs 22.2 u/10(6) cells at 72 hr). These findings suggest that anti-TNF antibody treatment may not only neutralize TNF activity, but also suppress TNF production itself, providing a new insight into the regulation of TNF by anti-TNF antibody.

Animals↗

PubMed1994

Alterations of K-ras, p53, and erbB-2/neu in human lung adenocarcinomas.

The development of human adenocarcinoma of the lung involves multiple genetic changes including activation of oncogenes and loss of tumor suppressor genes. Patients whose lung tumors contain K-ras oncogene mutation, accumulation of the protein product of the tumor suppressor gene p53, or erbB-2/neu oncoprotein overexpression have been shown to have a worse prognosis. We examined these three genetic indicators in 29 lung adenocarcinomas to determine whether these markers are present in the same tumors or if they represent molecular changes that define different subsets of patients. P53 nuclear protein accumulation and erbB-2/neu protein overexpression were determined by immunohistochemical analysis of cryostat sections of tumor specimens and corresponding normal lung tissue. K-ras mutations were detected by radiolabeled oligonucleotide probes, specific for the various twelfth codon mutations, hybridized to exon 1 of K-ras, which was amplified by the polymerase chain reaction. Increased nuclear accumulation of p53 protein was found in 11 adenocarcinomas (38%). All of the p53 positive tumors were found to show high level staining and homogeneous expression of erbB-2/neu protein. K-ras mutations were detected in seven tumors (24%), all of which overexpressed erbB-2/neu. The presence of a K-ras mutation did not correlate with p53 accumulation. In total, 93% of the tumors were found to overexpress erbB-2/neu, the highest being in one tumor with erbB-2/neu gene amplification. The presence of K-ras twelfth codon mutation was associated with increased cigarette smoking. In conclusion, erbB-2/neu overexpression is a common event in lung adenocarcinomas. Furthermore, the presence of K-ras mutation and p53 protein accumulation define separate groups of patients. The mechanisms by which these genetic alterations interact or adversely affect prognosis is unknown.

Adenocarcinoma↗

PubMed1993

Amplification and over-expression of the EGFR and erbB-2 genes in human esophageal adenocarcinomas.

Esophageal cancer is one of the 10 most prevalent human cancers worldwide. The incidence of esophageal adenocarcinoma is on the rise and patients with this disease typically have very poor prognosis. Informative biomarkers would benefit the clinical management of this disease. We examined 13 cases with esophageal adenocarcinomas and 5 cases with Barrett's esophagus for amplification of the EGFR and erbB-2 genes. We detected multiple copies of the EGFR gene in 30.8% of the tumors and multiple copies of the erbB-2 gene in 15.4% of the tumors. Of the cases with amplification of the erbB-2 gene, co-amplification of the EGFR gene was found. Multiple copies of the EGFR gene were also found in one case of Barrett's esophagus. Immunohistochemical staining of the tissues revealed increased expression of the erbB-2 protein in Barrett's mucosa and adenocarcinoma, but no associations between staining intensity and degree of EGFR or erbB-2 gene amplification, histology, or tumor stage were found. Differential polymerase chain reaction was examined as a method for pre-operative detection of gene amplification in esophageal tumors and Barrett's mucosa.

Adenocarcinoma↗

PubMed1993

Altered dexamethasone responsiveness and loss of growth control in tumorigenic mouse lung cell lines.

Glucocorticoid hormones induce differentiation, inhibit proliferation, and, in mice, reduce carcinogen-induced tumorigenesis of lung epithelial cells. Therefore we examined dexamethasone effects on tumorigenic and non-tumorigenic mouse lung epithelial-derived cell lines. Non-tumorigenic cells were growth inhibited and exhibited CAT activity in pMMTV-CAT transfectants in response to dexamethasone. Tumorigenic cell lines exhibited a range of responses to dexamethasone. While one tumorigenic line was growth-inhibited and responsive in CAT assays, 2 other tumorigenic cell lines were unresponsive both in CAT and in growth assays. A fourth tumorigenic cell line exhibited intermediate sensitivity in CAT assays and was actually growth-enhanced by dexamethasone. Although no difference between cell lines was observed in the abundance of glucocorticoid receptor protein on Western blots, the least dexamethasone-responsive tumorigenic lines exhibited very little binding of 3H-dexamethasone. Clones of tumorigenic lines stably transfected with the rat glucocorticoid receptor gene were more dexamethasone-sensitive in CAT assays and were growth-inhibited by dexamethasone. These data suggest that the neoplastic progression of cell lines derived from mouse lung frequently involves the acquisition of diminished glucocorticoid responsiveness.

Animals↗

PubMed1993

Reduction of hepatic phosphoenolpyruvate carboxykinase (PEPCK) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is due to decreased mRNA levels.

We have previously shown that the rate of hepatic gluconeogenesis is reduced in TCDD-treated rats and that this decrease in carbohydrate production is associated with a dose-dependent reduction of the activity of PEPCK, the rate limiting enzyme of gluconeogenesis. This derailment of glucose metabolism has been suggested to be the critical lesion in acute TCDD toxicity. To further elucidate the mechanism of decreased PEPCK activity we performed Northern blot analyses using a cDNA probe complementary to a portion of the mRNA coding for PEPCK. We have demonstrated that 4 and 8 days after TCDD treatment (125 micrograms/kg, p.o.) liver PEPCK mRNA in Sprague-Dawley rats was decreased to very low levels as compared to vehicle-treated and pair-fed control animals. This decline of PEPCK mRNA was paralleled by decreased levels of PEPCK protein, as revealed by Western blot analyses and was accompanied by a reduction in the enzymatic activity of PEPCK. These results indicate that the decrease of PEPCK activity by TCDD is most likely the result of decreased expression of the PEPCK gene. These together with previous results also suggest that many of the physiological responses occurring in TCDD-treated animals (reduced feed intake, decreased insulin, increased corticosterone, increased glucagon and cAMP levels) which would normally stimulate PEPCK gene expression, are ineffective. Furthermore tryptophan 2,3-dioxygenase (TdO) activity, which is regulated in a very similar fashion to PEPCK activity, is also reduced after TCDD treatment, suggesting a common mechanism by which TCDD alters the regulation of these enzymes. P-450 1A1 mRNA and related EROD activity were maximally induced under the conditions of these experiments and represent a positive control for TCDD-related alterations of gene expression. However, because of differences in the dose-response characteristics of TCDD-induced reduction of PEPCK activity and induction of EROD activity an involvement of the Ah receptor in the reduction of PEPCK activity cannot be postulated.

Animals↗

PubMed1993

Ki-ras mRNA regulation in untransformed mouse lung cells.

Although the Ki-ras gene is an often-observed transforming gene in lung tumors, little is understood of the factors that regulate the expression of the normal gene in lung cells. Therefore, we used untransformed mouse lung epithelial cells to determine the effect of serum, growth factors, and cell confluence on the regulation of Ki-ras mRNA expression. In subconfluent cells synchronized by 24-h serum deprivation, the refeeding of media containing serum resulted in the expression of both Ki-ras and H4 histone mRNA. No change in the expression of either gene was observed in cells refed with media alone. In confluent cell cultures, the refeeding of media with serum had no effect on the expression of these genes, suggesting that cell-density-dependent mechanisms can override the serum-induced stimuli for Ki-ras and H4 histone mRNA expression. Confluent cells expressed low but detectable Ki-ras mRNA levels consistent with constitutive expression of this gene independent of its role in mitogenic stimuli. EGF (10 ng/mL) and TGF-alpha (10 ng/mL) were found to induce transient increases in Ki-ras mRNA but large increases in H4 histone mRNA levels. Similarly, 48-h-conditioned media obtained from two transformed mouse lung cells containing activated Ki-ras genes and overexpressing Ki-ras mRNA were observed to increase both Ki-ras and H4 histone mRNA in the untransformed mouse lung cells. The expression of these genes in mouse lung cells therefore appeared to be linked to stimuli provided by specific growth factors as well as by autocrine factors elaborated by lung tumor cells. The studies reported here provide insight into the regulation of the Ki-ras mRNA in untransformed lung cells and the factors that may contribute to the elevated levels of Ki-ras mRNA often observed in transformed lung cells.

Animals↗