Expression of human gastrin gene in normal and gastrinoma tissues
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Cited by 31 publications
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Background. The amplification and/or overexpression of the HER‐2/neu oncogene has been proposed as an important prognostic marker in breast cancer. However, contradictory results from various groups regarding whether there is statistical significance in HER‐2 amplification or overexpression in predicting overall and disease free survival in node positive versus node negative patients exist in the literature. Current assays on quantifying the HER‐2 oncogene rely on DNA extracted from homogenized breast tissue. Not only is a large amount of tissue required, but also, the DNA extract is contaminated with DNA from stromal cells and leukocytes, leading to decreased specificity and sensitivity of the HER‐2 assay. Improving the specificity (DNA from breast ductal cells) and the sensitivity (competitive polymerase chain reaction [PCR]) of the HER‐2 amplification detection assay will help resolve some of these controversies. Methods. Using multiparameter flow cytometry (FCM), ductal cells from breast biopsies and fine needle aspirations (FNAs) are identified and selectively sorted using anti‐cytokeratin, anti‐HER‐2 antibody labeling and DNA staining. HER‐2 amplification in these sorted cells is then quantified by competitive DNA PCR using a competitive reference standard mutant template that is susceptible to the restriction enzyme Sma‐1. Results. Applying this strategy, SK‐BR‐3, an HER‐2 amplified breast cancer cell line, was found to have approximately 9X baseline HER‐2 oncogene copies. In addition, MCF‐7, a known HER‐2 nonamplified breast cancer cell line, was found to have baseline HER‐2 oncogene copies. In the 10 clinical breast samples tested, 4 of the 10 breast cancers were HER‐2 amplified using as few as 1000 cells. The cytokeratin positive cells of these cancers, in contrast to the cytokeratin negative cells, have detectably higher HER‐2 amplification (7.2 ± 2.8X versus 3.2 ± 1.1X, respectively). Hence, HER‐2 gene amplification would have been underestimated if unsorted cells were used because of stromal dilution. In the cytokeratin positive cells that were HER‐2 oncogene amplified, corresponding HER‐2 oncoprotein overexpression was detected by FCM. Conclusions. Using FCM, the ductal cell subpopulation of a breast specimen can be successfully sorted from breast biopsy and FNA specimens. Moreover, by applying the technique of competitive PCR, improved specificity and sensitivity in HER‐2 oncogene amplification detection is achieved. The entire procedure can be accomplished in 1 day, allowing for a cost‐effective assay and rapid turnaround time. Cancer 1994; 73:2771–8.
Background. The amplification and/or overexpression of the HER‐2/neu oncogene has been proposed as an important prognostic marker in breast cancer. However, contradictory results from various groups regarding whether there is statistical significance in HER‐2 amplification or overexpression in predicting overall and disease free survival in node positive versus node negative patients exist in the literature. Current assays on quantifying the HER‐2 oncogene rely on DNA extracted from homogenized breast tissue. Not only is a large amount of tissue required, but also, the DNA extract is contaminated with DNA from stromal cells and leukocytes, leading to decreased specificity and sensitivity of the HER‐2 assay. Improving the specificity (DNA from breast ductal cells) and the sensitivity (competitive polymerase chain reaction [PCR]) of the HER‐2 amplification detection assay will help resolve some of these controversies. Methods. Using multiparameter flow cytometry (FCM), ductal cells from breast biopsies and fine needle aspirations (FNAs) are identified and selectively sorted using anti‐cytokeratin, anti‐HER‐2 antibody labeling and DNA staining. HER‐2 amplification in these sorted cells is then quantified by competitive DNA PCR using a competitive reference standard mutant template that is susceptible to the restriction enzyme Sma‐1. Results. Applying this strategy, SK‐BR‐3, an HER‐2 amplified breast cancer cell line, was found to have approximately 9X baseline HER‐2 oncogene copies. In addition, MCF‐7, a known HER‐2 nonamplified breast cancer cell line, was found to have baseline HER‐2 oncogene copies. In the 10 clinical breast samples tested, 4 of the 10 breast cancers were HER‐2 amplified using as few as 1000 cells. The cytokeratin positive cells of these cancers, in contrast to the cytokeratin negative cells, have detectably higher HER‐2 amplification (7.2 ± 2.8X versus 3.2 ± 1.1X, respectively). Hence, HER‐2 gene amplification would have been underestimated if unsorted cells were used because of stromal dilution. In the cytokeratin positive cells that were HER‐2 oncogene amplified, corresponding HER‐2 oncoprotein overexpression was detected by FCM. Conclusions. Using FCM, the ductal cell subpopulation of a breast specimen can be successfully sorted from breast biopsy and FNA specimens. Moreover, by applying the technique of competitive PCR, improved specificity and sensitivity in HER‐2 oncogene amplification detection is achieved. The entire procedure can be accomplished in 1 day, allowing for a cost‐effective assay and rapid turnaround time. Cancer 1994; 73:2771–8.
1Tyrosine 0-sulfation is a common post-translational modification of secretory and membrane proteins. The biological function of sulfation is known in only a few proteins, where it appears to enhance protein-protein interactions. Based on known sequences around sulfated tyrosines, a consensus sequence for prediction of target tyrosines has been proposed. However, some proteins are tyrosine sulfated at sites that deviate from the proposed consensus. Among these is progastrin. It is possible that the deviation explains the incomplete sulfation characteristic for bioactive gastrin peptides. In order to test this hypothesis, we have performed site-directed mutagenesis of the gastrin gene followed by heterologous expression in an endocrine cell line. The results show that substitution of the alanyl residue immediately N-terminal to the sulfated tyrosine with an acidic amino acid promotes the sulfation of gastrin peptides. Hence, the study supports the proposed consensus sequence for tyrosine sulfation. Importantly, however, the results also reveal that complete sulfation increases the endoproteolytic maturation of progastrin. Thus, our study suggests an additional function for tyrosine sulfation of possible general significance.
Gastrin is transcriptionally responsive to EGF stimulation (Merchant et al., 1991, Mol. Cell. Biol., 11:2686-2696). Consequently, we hypothesized that previously recognized gastrin autocrine loops (Hoosein et al., 1990, Exp. Cell. Res., 186:15-21), might be controlled by autocrine TGF alpha in human colon carcinoma cells. Therefore, we examined the interaction between these two autocrine growth factors in two colon carcinoma cell lines which utilize TGF alpha. The FET cell line requires exogenous TGF alpha/EGF for optimal growth and has a classical TGF alpha autocrine loop which is disrupted by TGF alpha or epidermal growth factor receptor (EGFr) antibodies. The HCT 116 cell line is not dependent on exogenous TGF alpha/EGF and exhibits a nonclassical TGF alpha autocrine loop which is not disrupted by neutralizing antibodies to either TGF alpha itself or the EGFr. Basal gastrin mRNA production is significantly higher in HCT 116 than FET as measured by RNase protection assay. In the FET cells, exogenous EGF stimulates gastrin mRNA production but not in HCT 116. When the TGF alpha autocrine loop in HCT 116 is disrupted by constitutive expression of antisense TGF alpha mRNA, the gastrin mRNA level is significantly repressed. In xenografts derived from these antisense clones, TGF alpha reverted to high expression, and the gastrin mRNA level was again increased. This interaction between the strong TGF alpha loop in HCT 116 and the gastrin autocrine loop may confer a growth advantage to these colon cells. Such interactions between growth factors may promote enhanced tumorigenicity to transformed cells with these strong, nonclassical autocrine loops.
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