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C W Schweinfest

Publications and source records attributed to C W Schweinfest.

At least 19 recordsLinked to original sources
PubMed2001

Defects of DNA mismatch repair in human prostate cancer.

Loss of mismatch repair (MMR) function leads to the accumulation of errors that normally occur during DNA replication, resulting in genetic instability. Germ-line mutations of MMR genes in the patients with hereditary nonpolyposis colorectal cancer lead to inactivation of MMR protein functions, and the defects of MMR are well correlated to the high rate of microsatellite instability in their tumors. Previous studies (T. Uchida, et al. Oncogene, 10: 1019-1022, 1995; S. Egawa, et al. Cancer RES:, 55: 2418-2421, 1995; J. M. Cunningham, et al. Cancer RES:, 56: 4475-4482, 1996; X. Gao, et al. Oncogene, 9: 2999-3003, 1994; H. Rohrbach, et al. Prostate, 40: 20-27, 1999) have shown that genetic instability (chromosomal and microsatellite instability) is detectable in human prostate cancer. To elucidate the role of MMR genes in the tumorigenesis of prostate cancer, we evaluated the expression of these genes in human cancer cell lines and in tumor specimens. Using Western blot analysis, we detected loss among MSH2, MLH1, PMS2, and PMS1 proteins in DU145, LNCaP, p69SV40T, M2182, and M12 cells. In addition, genomic instability in the prostate cell lines including DU145, PC3, LNCaP, p67SV40T, M2182, and M12 was detected by a microsatellite mutation assay. Significantly, immunohistochemical analysis of prostatic tissue revealed the reduction or absence of MMR protein expression in the epithelium of prostate tumor foci compared with normal adjacent prostate tissue. In contrast to hereditary nonpolyposis colorectal cancer, characterized by defects predominantly in MLH1 and MSH2, the samples we examined showed more tumor foci with loss of PMS1 and PMS2. PMS1, which is only expressed in the basal cells in normal glands, is conspicuously absent in most prostate cancer. From these results, we conclude that there are defects of MMR genes in human prostate cancer.

Adaptor Proteins, Signal Transducing↗
PubMed2001

CaSm/gemcitabine chemo-gene therapy leads to prolonged survival in a murine model of pancreatic cancer.

BACKGROUND: CaSm, the cancer-associated Sm-like oncogene, is overexpressed in greater than 80% of pancreatic tumors. We previously reported that an adenovirus expressing antisense RNA to CaSm (Ad-alpha CaSm) can decrease pancreatic tumor growth in vivo but is not curative. In the current study we investigated the mechanism of Ad-alpha CaSm's antitumor effect to rationally approach combinatorial therapy for improved efficacy. METHODS: AsPC-1 and Panc-1 human pancreatic cancer cells were treated with Ad-alpha CaSm and examined by MTT assay for in vitro proliferation changes. Flow cytometry determined the effect of CaSm down-regulation on the cell cycle, and then cells treated with Ad-alpha CaSm in combination with cisplatin, etoposide, or gemcitabine chemotherapies were reexamined by MTT assay. SCID-Bg mice bearing subcutaneous AsPC-1 tumors were treated with Ad-alpha CaSm, gemcitabine, or the combination and monitored for tumor growth and survival. RESULTS: Treatment with Ad-alpha CaSm reduced the proliferation of AsPC-1 and Panc-1 cells (59% and 44%, respectively; P <.05). The cell cycle revealed a cytostatic block with decreased G(1) phase and increased DNA content in treated cells. The combination of Ad-alpha CaSm with gemcitabine significantly reduced in vitro proliferation (66% vs 39% and 48% for controls), decreased in vivo AsPC-1 tumor growth by 71% (n = 10), and extended survival time from 57 to 100 days. CONCLUSIONS: Down-regulation of CaSm reduces the growth of pancreatic cancer cells by altering the cell cycle in a cytostatic manner. The combination of Ad-alpha CaSm with gemcitabine is more effective than either agent used separately.

Animals↗
PubMed2001

Downregulated in adenoma and putative anion transporter are regulated by CFTR in cultured pancreatic duct cells.

The mechanism of the pancreatic ductal HCO secretion defect in cystic fibrosis (CF) is not well defined. However, a lack of apical Cl(-)/HCO exchange may exist in CF. To test this hypothesis, we examined the expression of Cl(-)/HCO exchangers in cultured pancreatic duct epithelial cells with physiological features prototypical of CF [CFPAC-1 cells lacking a functional CF transmembrane conductance regulator (CFTR)] or normal duct cells (CFPAC-1 cells transfected with functional wild-type CFTR, CFPAC-WT). Cl(-)/HCO exchange activity, assayed with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in cells grown on coverslips, increased about twofold in cells transfected with functional CFTR. This correlated with increased apical (36)Cl influx in cells expressing functional CFTR and grown on permeable support. Northern hybridizations indicated the induction of downregulated in adenoma (DRA) in cells expressing functional CFTR. The expression of putative anion transporter PAT1 also increased significantly in cells expressing functional CFTR. DRA was detected at high levels in native mouse pancreas by Northern hybridization and localized to the apical domain of the duct cells by immunohistochemical studies. In conclusion, CFTR upregulates DRA and PAT1 expression in cultured pancreatic duct cells. We propose that the pancreatic HCO secretion defect in CF patients is partly due to the downregulation of apical Cl(-)/HCO exchange activity mediated by DRA (and possibly PAT1).

Animals↗
PubMed2000

The cancer-associated Sm-like oncogene: a novel target for the gene therapy of pancreatic cancer.

BACKGROUND: The prognosis for pancreatic cancer (PC) remains dismal, providing a clear need for the development of novel therapies. We have previously shown that the cancer-associated Sm-like (CaSm) oncogene is overexpressed in the great majority of pancreatic tumors and is required to maintain the transformed phenotype. The purpose of this study was to determine whether the application of CaSm antisense gene therapy would generate a significant antitumor effect against PC. METHODS: An adenoviral vector (Ad-alphaCaSm) expressing a 900-base pair antisense RNA to CaSm was created. The PC cell lines AsPC-1 and Capan-1 were infected with this vector and examined for changes in in vitro proliferation by using methyl thiazol tetrazolium and soft agar assays. SCID-Bg mice bearing subcutaneous AsPC-1 tumors were treated with Ad-alphaCaSm (1 x 10(9) plaque-forming units) as a single intratumor injection with tumor growth and survival monitored. RESULTS: AsPC-1 and Capan-1 cells showed decreased in vitro proliferation (93%, P =.0041, and 70%, P =. 0038, respectively) and anchorage independent growth (55%, P =.02, and 45%, P =.03, respectively) after treatment. Ad-alphaCaSm reduced in vivo AsPC-1 tumor growth by 40% (n = 10), extending median survival time from 35 to 60 days. CONCLUSIONS: Ad-alphaCaSm demonstrates a significant antitumor effect against pancreatic cancer both in vitro and in vivo. These results support the role of CaSm as a significant gene involved in the neoplastic transformation of pancreatic tumors. Thus CaSm represents a novel gene target in PC and holds potential as a new treatment approach either alone or in combination with existing therapies.

Adenoviridae↗
PubMed1999

H,K-ATPase alpha subunit C-terminal membrane topology: epitope tags in the insect cell expression system.

The H,K-ATPase responsible for gastric acidification is a heterodimeric (alpha and beta subunit) P-type ATPase, an integral protein of parietal cell apical membranes, which promotes the electroneutral exchange of K+ for protons, is stimulated by K+ and is inhibited by 2-methyl-8-(phenylmethoxy)imidazo[1, 2-alpha]pyridine-3-acetonitrile (SCH 28080). Hydropathy analysis of the catalytic alpha subunit has been interpreted in terms of four N-terminal transmembrane domains, a cytoplasmically oriented segment containing ATP binding and phosphorylation sites, and a C-terminal region with four or six putative transmembrane domains. Several lines of evidence implicate the C-terminal region of P-type ATPases in cation-binding and occlusion, conformational changes, and interactions with the beta subunit (HKbeta), making the definition of topology a prerequisite for understanding the structural basis of these functions. Influenza haemagglutinin epitopes (YPYDVPDYA; flu tag) were inserted in predicted hydrophilic segments of the alpha subunit (HKalpha) to establish the membrane orientation of two amino acids with different predicted topologies in the C-terminal four- and six-transmembrane models. Wild-type and mutated HKalpha and HKbeta cDNA species were expressed in insect cells (Sf9) via recombinant baculovirus infection, and expression of H,K-ATPase was verified by immunoblotting with HKalpha- and HKbeta-specific and flu-tag-specific antibodies. Functional assays showed K+-stimulated, SCH 28080-sensitive ATPase activity, confirming neo-native topology in H,K-ATPase heterodimers expressed in Sf9 cells. The topology of flu tags was determined by microsomal protease protection assays in Sf9 cells and immunolabelling of HKalpha and HKbeta in intact and permeabilized Sf9 cells. In addition, MS of native H,K-ATPase tryptic peptides identified cytoplasmically oriented HKalpha residues. The results indicated cytoplasmic exposure of Leu844 and Phe996, and luminal exposure of Pro898, leading to a revised secondary structure model of the C-terminal third of HKalpha.

Adenosine Triphosphate↗
PubMed1998

ETS2 function is required to maintain the transformed state of human prostate cancer cells.

The contribution of the ETS2 transcription factor to the transformed state in prostate cancer cells has been assessed. Northern blot analysis easily detects ETS2 in DU145 and PC3, high grade human prostate cell lines, but ETS2 is not present in lower grade LNCaP cells. Stable transfection of PC3 and DU145 prostate cell lines with an antisense ETS2 vector or with a dominant negative ETS2 mutant significantly reduced the ability of DU145 and PC3 cells to form large colonies in soft agar. Thus, the presence of ETS2 is positively correlated with a more transformed phenotype and blockage of ETS2 function can reduce transformed properties of prostate cancer cells.

Agar↗
PubMed1998

Cloning and sequence analysis of Hsp89alpha DeltaN, a new member of theHsp90 gene family.

We have identified a novel member of the Hsp90 gene family. This new gene, Hsp89alpha DeltaN, is remarkable in that it appears to represent a recent evolutionary event. Hsp89alpha DeltaN is identical in nucleotide sequence to Hsp89alpha for codons 224 to 732 (end). However, Hsp89alpha DeltaN cDNA lacks the ATP/geldanamycin binding domain (codons 1-220), instead containing 544 nucleotides of unique DNA at its 5' end including 30 novel codons.

Amino Acid Sequence↗
PubMed1998

Human DRA functions as a sulfate transporter in Sf9 insect cells.

DRA is a gene that is down-regulated in colon adenomas and adenocarcinomas in humans. We have previously shown that DRA proteins are found as various forms in tissue due to differential glycosylation. This study has focused on the function of DRA related to its subcellular localization. We used the baculovirus expression system and overexpressed a nearly full-length DRA driven by a polyhedrin promoter in Sf9 insect cells. DRA protein expressed in this cell was underglycosylated relative to normal colon mucosa, but uniformly targeted to the cell membrane. It also appears to undergo posttranslational cleavage, removing about 100 amino acids from its amino terminus. This membrane localization is similar to what we observed in the colon mucosa. An ion transport assay demonstrated that DRA functions as a sulfate transporter. When DRA was expressed, sulfate import was increased more than threefold compared to the control. Sulfate import was inhibitable by the anion transporter inhibitor, DIDS, in a dose-dependent fashion. Given that (1) DRA has high similarity to other identified sulfate transporters and the proposed structure of DRA polypeptide is characteristic of those transporters, (2) DRA localization is limited to the cell membrane, and (3) DRA expression correlates with intestinal differentiation in mouse, we suggest that DRA represents a tissue-specific member of the sulfate transporter family.

Amino Acid Sequence↗
PubMed1998

Intestinal inflammation reduces expression of DRA, a transporter responsible for congenital chloride diarrhea.

The pathogenesis of diarrhea in intestinal inflammatory states is a multifactorial process involving the effects of inflammatory mediators on epithelial transport function. The effect of colonic inflammation on the gene expression of DRA (downregulated in adenoma), a chloride-sulfate anion transporter that is mutated in patients with congenital chloridorrhea, was examined in vivo as well as in an intestinal epithelial cell line. DRA mRNA expression was diminished five- to sevenfold in the HLA-B27/beta2m transgenic rat compared with control. In situ hybridization showed that DRA, which is normally expressed in the upper crypt and surface epithelium of the colon, was dramatically reduced in the surface epithelium of the HLA-B27/beta2m transgenic rat, the interleukin-10 (IL-10) knockout mouse with spontaneous colitis, and in patients with ulcerative colitis. Immunohistochemistry demonstrated that mRNA expression of DRA reflected that of protein expression in vivo. IL-1beta reduced DRA mRNA expression in vitro by inhibiting gene transcription. The loss of transport function in the surface epithelium of the colon by attenuation of transporter gene expression, perhaps inhibited at the level of gene transcription by proinflammatory cytokines, may play a role in the pathogenesis of diarrhea in colitis.

Animals↗
PubMed1998

Down-regulation of the down-regulated in adenoma (DRA) gene correlates with colon tumor progression.

The down-regulated in adenoma (DRA) gene was originally identified as a gene that was down-regulated in colon tumors. It encodes a protein with anion transporter function that is expressed predominantly in the mucosa of the lower gastrointestinal tract. In this study, expression of DRA and its cellular distribution have been investigated in a series of benign adenomatous polyps and malignant colorectal tumors and in corresponding normal colonic mucosa. We show that DRA mRNA and protein are expressed in all normal colonic tissue specimens with the protein restricted primarily to the terminally differentiated columnar epithelium and some goblet cells. Apical membrane localization was especially apparent in the columnar epithelium. The levels of DRA mRNA transcripts were down-regulated in all colon tumors examined relative to matched normal mucosa, with most specimens showing undetectable levels of DRA mRNA (77 of 104 tumors). DRA down-regulation was positively associated with colonic tumor progression according to Dukes' stage and was particularly significant in the early transition from normal mucosa to polyp to adenocarcinoma. DRA expression does not appear to be strictly associated with colonic cell differentiation; rather, its absence and down-regulation were associated with the proliferating component of the crypt epithelium and with neoplastic transformation, respectively.

Adenoma↗
PubMed1997

Characterization of human N8 protein.

We have shown before that the N8 mRNA is expressed at higher levels in lung tumor and lung tumor-derived cell lines than normal lung cells. In this paper, we have characterized the N8 protein, and studied its properties. The N8 gene encodes a major 24 kDa protein and its expression correlates well with the N8 mRNA expression pattern observed in different cell lines. N8 protein is capable of forming a homodimer or multimeter in vitro. It is a phosphorylated cytoplasmic protein and phosphorylation occurs mainly at serine residues. N8 protein is expressed at higher levels in epithelial cells than in mesenchymal cells. N8 protein expression is induced in a fibroblast cell line expressing adenoviral Ela protein, which acquired epithelial-like characteristics. Furthermore, ectopic expression of N8 protein in NIH3T3 cells converts them into a spheroid form. These spheroids also have some of the characteristic features of epithelial cells. Taken together, these results suggest that the N8 protein may be associated with the development or maintenance of epithelial cell phenotype.

3T3 Cells↗
PubMed1997

CaSm: an Sm-like protein that contributes to the transformed state in cancer cells.

A novel gene encoding a protein containing Sm motif-like domains was found to have elevated expression in pancreatic cancer and in several cancer-derived cell lines. CaSm (for Cancer-associated Sm-like) mRNA is up-regulated in 87.5% (seven of eight) of pancreatic tumor/normal pairs. Similarly, cell lines from cancers originating in liver, ovary, lung, and kidney show increased CaSm expression compared to their normal tissue cognates. CaSm encodes a 133-amino acid open reading frame that contains the two Sm motifs found in the common snRNP proteins, with the greatest homology to the Sm G protein (60% similarity). Two hypothetical proteins from Caenorhabditis elegans and Saccharomyces cerevisiae share even greater similarity (72.8 and 67.7%, respectively), suggesting a broad family of proteins containing Sm motifs. Antisense CaSm RNA is able to alter the transformed phenotype of pancreatic cancer cells by reducing their ability to form large colonies in soft agar when compared to untransfected cells. Therefore, CaSm expression appears to be necessary for maintenance of the transformed state.

Amino Acid Sequence↗
PubMed1996

The S29 ribosomal protein increases tumor suppressor activity of K rev-1 gene on v-K ras-transformed NIH3T3 cells.

The human S29 ribosomal protein (S29 rp) cDNA has been isolated from differential hybridization screening of a colon carcinoma cDNA library. Northern blot analysis showed that the level of S29 rp mRNA was higher in undifferentiated HT29 human colon carcinoma cells than in a morphologically differentiated subclone under the same growth condition. Furthermore, the level of S29 rp mRNA was downregulated in rapidly proliferating HT29 cells, as compared to the contact inhibited cells. Interestingly, the amount of Krev-1 mRNA was inversely correlated with respect to the amount of S29 rp mRNA in these cells. To examine a functional link between S29 rp and Krev-1 protein, we co-transfected the expression vectors containing wild-type or mutant S29 rp and mutationally activated Krev-1(63E) cDNAs into the v-Ki-ras-transformed NIH3T3 (DT) cells, and observed the induction of flat revertants. Krev-1(63E) induced a certain amount of flat colonies, while S29 rp alone also induced flat colonies at low frequencies. Interestingly, revertant-inducing activity of Krev-1(63E) was significantly enhanced by S29 rp. We have also demonstrated that a zinc finger-like domain of S29 rp indeed has a zinc binding activity and a derivative, S29 rp(ms), which was unable to bind zinc ion but still retained revertant inducing activity by itself, could not functionally interact with Krev-1(63E) protein.

3T3 Cells↗
PubMed1996

The down-regulated in adenoma (DRA) gene encodes an intestine-specific membrane glycoprotein.

The protein product of the DRA gene, a gene whose expression is down-regulated in colon adenomas and adenocarcinomas, is a membrane glycoprotein and a member of a family of sulfate transporters. It is expressed in the intestinal tract (duodenum, ileum, cecum, distal colon), but not in the esophagus or stomach. DRA mRNA expression is restricted to the mucosal epithelium, and DRA protein expression is further limited to the columnar epithelial cells, particularly to the brush border. Consistent with its expression in the differentiated columnar epithelium of the adult human colon, DRA is first expressed in the midgut of developing mouse embryos at day 16.5, corresponding with the time of differentiation of the epithelium of the small intestine. A model for the structure of the DRA protein is proposed and its possible role in colon tumorigenesis is discussed.

Animals↗
PubMed1996

Isolation of differentially expressed genes in carcinoma of the esophagus.

BACKGROUND: The genetic alterations that occur in the transformation of normal esophageal mucosa (NEM) to carcinoma of the esophagus (CAE) are not well understood. Differential display of mRNA is a recently described technique that uses reverse transcription and PCR to compare cDNA from paired normal and malignant tissue to determine whether there is either genetic loss (putative tumor suppressor gene) or overexpression (putative oncogene) in malignant cells. Our goal was to identify some of these genes from patients with CAE. METHODS: Specimens of NEM and corresponding CAE were obtained from patients at endoscopy or surgical resection and immediately snap frozen. Total RNA was isolated, reverse transcribed to cDNA, and PCR amplified with a predefined 10-mer oligonucleotide. The products were displayed on a polyacrylamide gel. Differential bands were isolated and sequenced and/or used as probes for Northern analysis. RESULTS: Application of the differential display method resulted in the isolation of 49 cDNA clones from three patients with CAE. Sequencing of the clones has revealed five unique sequences not previously reported and one that has been identified as histone H3.3. Northern analysis of histone H3.3 has revealed overexpression in four of six CAEs but not the paired NEM. In addition, whereas only 5 of 13 normal human cell lines of various origins overexpressed this gene, 11 of 12 human cancer cell lines (9 of 9 adenocarcinomas) overexpressed it. CONCLUSIONS: Differential display can be used to isolate potential oncogenes and tumor suppressor genes. We have identified five unique sequences and one known gene that may contribute to the development of CAE.

Adenocarcinoma↗
PubMed1993

Identification of a colon mucosa gene that is down-regulated in colon adenomas and adenocarcinomas.

A cDNA, which we call DRA (for down-regulated in adenoma) has been isolated. Its mRNA is expressed exclusively in normal colon tissue, probably only in the mucosal epithelia. Expression of the DRA gene is significantly decreased in adenomas (polyps) and adenocarcinomas of the colon. The DRA gene appears to be a single-copy gene present on chromosome 7, a chromosome associated with colorectal tumorigenesis. The predicted DRA polypeptide is an 84,500-Da protein that contains charged clusters of amino acids, primarily at the NH2 and COOH termini. Together with potential nuclear targeting motifs, an acidic transcriptional activation domain, and a homeobox domain, these elements suggest a transcription factor or a protein that may interact with transcription factors. Such a function may be consistent with a role in tissue-specific gene expression and/or as a candidate tumor-suppressor gene.

Adenocarcinoma↗