A novel autosomal dominant limb-girdle muscular dystrophy (LGMD 1F) maps to 7q32.1-32.2.

In 2001, the authors described the clinical features of a genetically distinct autosomal dominant limb-girdle muscular dystrophy (LGMD; LGMD 1F). Using a genome-wide screen with more than 400 microsatellite markers, the authors identified a novel LGMD disease locus at chromosome 7q32.1-32.2. Within this chromosomal region, filamin C, a gene encoding actin binding protein highly expressed in muscle, was an obvious candidate gene; however, the authors did not detect any defects in filamin C or its protein product.

Reversion of mtDNA depletion in a patient with TK2 deficiency.

Mutations in the thymidine kinase 2 (TK2) gene cause a myopathic form of the mitochondrial DNA depletion syndrome (MDS). Here, the authors report the unusual clinical, biochemical, and molecular findings in a 14-year-old patient in whom pathogenic mutations were identified in the TK2 gene. This report extends the phenotypic expression of primary TK2 deficiency and suggests that factors other than TK2 may modify expression of the clinical phenotype in patients with MDS syndrome.

Defects of intergenomic communication: autosomal disorders that cause multiple deletions and depletion of mitochondrial DNA.

Depletion and multiple deletions of mitochondrial DNA (mtDNA) have been associated with a growing number of autosomal diseases that have been classified as defects of intergenomic communication. MNGIE, an autosomal recessive disorder associated with mtDNA alterations is due to mutations in thymidine phosphorylase that may cause imbalance of the mitochondrial nucleotide pool. Subsequently, mutations in the mitochondrial proteins adenine nucleotide translocator 1, Twinkle, and polymerase gamma have been found to cause autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA. Uncovering the molecular bases of intergenomic communication defects will enhance our understanding of the mechanisms responsible for maintaining mtDNA integrity.

Uncoupling protein-1 mRNA expression in lipomas from patients bearing pathogenic mitochondrial DNA mutations.

Multiple symmetric lipomatosis (MSL) is a rare disorder characterised by large subcutaneous fat masses in some parts of the trunk. Mitochondrial disfunction is common in MSL, but the identity of the adipose cells developing in multiple lipomas is not well known. We determined that brown adipose tissue-specific uncoupling protein-1 (UCP-1) mRNA is expressed in the lipoma of a multiple symmetric lipomatosis patient bearing the 8344 mutation in the tRNALys gene of mitochondrial DNA. UCP1 mRNA was not detected in normal subcutaneous fat from the same patient or in the lipoma of another patient bearing a different mutation in the same tRNALys gene. These findings implicate brown adipose cells as the origin of lipomas in a subset of patients bearing tRNALys mutations in mitochondrial DNA.

Increased glyceraldehyde-3-phosphate dehydrogenase expression in renal cell carcinoma identified by RNA-based, arbitrarily primed polymerase chain reaction.

BACKGROUND: Renal cell carcinoma (RCC) comprises 85% of renal tumors and displays a great capacity to metastasize. The lack of diagnostic and prognostic markers complicates its early detection and in the majority of cases metastases are present at the time of diagnosis. METHODS: The current study reports on the identification of differentially expressed genes in RCC using random arbitrarily primed polymerase chain reaction (RAP-PCR). RESULTS: Four genes were identified, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase A (LDH A), human leukocyte antigen A (HLA A), and ferritin. GAPDH and HLA A were found to be overexpressed in 100% of the tumors and LDH A was increased in > 85% of the tumors analyzed compared with normal kidney counterparts. For GAPDH and LDH A higher protein levels in the tumors also were determined by Western blot analysis. Differential expression did not appear to correlate with gene amplification events as demonstrated by Southern blot analysis, indicating that regulatory mechanisms controlling the expression of these genes were altered. Finally, ferritin was judged to have a variable expression because it was decreased in approximately 50% of the tumors and augmented in 20%. The implications in proliferation and differentiation of all these genes were analyzed in RCC cell lines grown at different stages of confluency and additional information was obtained regarding expression of the GAPDH gene in proliferating primary cultures of normal and tumor cells derived from the same kidney samples. CONCLUSIONS: The authors conclude that RAP-PCR is a useful technique with which to identify rapidly differentially expressed genes in a given system. In addition, they also conclude that GAPDH is a potent marker of cell proliferation in kidney tumor cells whose overexpression appears to be a late event in the development of RCC.

Activation of the urokinase plasminogen activator/urokinase plasminogen activator receptor system and redistribution of E-cadherin are associated with hepatocyte growth factor-induced motility of pancreas tumor cells overexpressing Met.

Because hepatocyte growth factor (HGF) is a potent mitogen for normal human exocrine pancreas cells (NPCs) in vitro, we have analyzed the expression of HGF and its receptor, Met, in NPC and pancreas cancer cells and studied its effects in vitro. Using immunohistochemistry, Northern blotting, and reverse transcription-polymerase chain reaction, we examined the expression of HGF and Met in normal pancreas and pancreas cancer. Scatter assays, wound-healing assays, and migration through transwell filters were used to study HGF-stimulated motility of IMIM-PC-2 cancer cells. In tumors, HGF is mainly detected in stromal cells, whereas Met is overexpressed in cancer cells with an unpolarized distribution. In vitro, HGF stimulates motogenesis but not proliferation in cancer cells. Cell motility is accompanied by a rapid decrease in the cytoskeleton-bound E-cadherin, an acceleration of cellular adhesion to the substrate, an up-regulation of urokinase plasminogen activator (u-PA) RNA and protein, and a change in the solubility and proteolysis of the u-PA receptor. Cell motility is significantly reduced by inhibitors of u-PA proteolytic activity such as antibodies neutralizing u-PA activity, plasminogen activator inhibitor 1, and amiloride. These results show that a paracrine loop of HGF activation may participate in the development or progression of pancreas cancer. In vitro, the HGF-stimulated motogenesis of pancreas cancer cells involves the activation of the u-PA/u-PA receptor proteolytic system, suggesting its role in the invasive stages of tumor progression.

Role of UEV-1, an inactive variant of the E2 ubiquitin-conjugating enzymes, in in vitro differentiation and cell cycle behavior of HT-29-M6 intestinal mucosecretory cells.

By means of differential RNA display, we have isolated a cDNA corresponding to transcripts that are down-regulated upon differentiation of the goblet cell-like HT-29-M6 human colon carcinoma cell line. These transcripts encode proteins originally identified as CROC-1 on the basis of their capacity to activate transcription of c-fos. We show that these proteins are similar in sequence, and in predicted secondary and tertiary structure, to the ubiquitin-conjugating enzymes, also known as E2. Despite the similarities, these proteins lack a critical cysteine residue essential for the catalytic activity of E2 enzymes and, in vitro, they do not conjugate or transfer ubiquitin to protein substrates. These proteins constitute a distinct subfamily within the E2 protein family and are highly conserved in phylogeny from yeasts to mammals. Therefore, we have designated them UEV (ubiquitin-conjugating E2 enzyme variant) proteins, defined as proteins similar in sequence and structure to the E2 ubiquitin-conjugating enzymes but lacking their enzymatic activity (HW/GDB-approved gene symbol, UBE2V). At least two human genes code for UEV proteins, and one of them, located on chromosome 20q13.2, is expressed as at least four isoforms, generated by alternative splicing. All human cell types analyzed expressed at least one of these isoforms. Constitutive expression of exogenous human UEV in HT-29-M6 cells inhibited their capacity to differentiate upon confluence and caused both the entry of a larger proportion of cells in the division cycle and an accumulation in G2-M. This was accompanied with a profound inhibition of the mitotic kinase, cdk1. These results suggest that UEV proteins are involved in the control of differentiation and could exert their effects by altering cell cycle distribution.

Cloning of a gene highly overexpressed in cancer coding for a novel KH-domain containing protein.

In a previous large scale screen for differentially expressed genes in pancreatic cancer, we identified a gene highly overexpressed in cancer encoding a novel protein with four K-homologous (KH) domains. KH-domains are found in a subset of RNA-binding proteins, including pre-mRNA-binding (hnRNP) K protein and the fragile X mental retardation gene product (FMR1). By fluorescence in situ hybridization (FISH) the identified gene named koc (KH domain containing protein overexpressed in cancer) was assigned to chromosome 7p11.5. Two pseudogenes were localised on chromosome 6 and 11. The cloned koc cDNA has a 250 bp 5'-UTR, a 1740 bp ORF and a 2168 bp 3'-UTR. The AU-rich 3'-untranslated region of koc contains eight AUUUA and four AUUUUUA reiterated motifs. The deduced koc protein with 580 amino-acids has a relative molecular mass (Mr) of approximately 65,000 (65 K). The koc transcript is highly overexpressed in pancreatic cancer cell lines and in pancreatic cancer tissue as compared to both, normal pancreas and chronic pancreatitis tissue. High levels of expression were as well found in tissue samples of other human tumours. As the KH domain has been shown to be involved in the regulation of RNA synthesis and metabolism, we speculate that koc may assume a role in the regulation of tumour cell proliferation by interfering with transcriptional and or posttranscriptional processes. However, the precise role of koc in human tumour cells is unknown and remains to be elucidated.

Hepatocyte growth factor is a potent mitogen for normal human pancreas cells in vitro.

BACKGROUND: Little is known regarding the mechanisms that regulate cell proliferation in the exocrine pancreas. The aim of this study was to analyze the effect of growth factors on normal human exocrine pancreas cultures. EXPERIMENTAL DESIGN: The mitogenic effect of hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and other growth factors was examined using [3H]-thymidine uptake assays. The phenotype of the cultures was analyzed using a panel of mAb. c-met expression was determined by immunohistochemistry and Western blotting. RESULTS: HGF was the most potent mitogen (concentration range 0.1 to 10 ng/ml), although IGF-1, EGF, and platelet-derived growth factor were also mitogenic in some assays. Combinations of HGF+EGF or HGF+IGF-1 were superior to HGF when used at suboptimal concentrations. Proliferative cultures expressed cytokeratins 7, 8, 18, and 19 and the MUC1 mucin, a phenotype characteristic of normal ductal cells. The 170-kDa precursor and 145-beta chain of c-met were detected in cultured cells with rabbit polyclonal antisera raised against synthetic peptides corresponding to the carboxy-terminal domains of the beta chain. c-met was also detected in the apical membrane of ductal cells in normal pancreas tissue but was generally absent from acinar, centroacinar, and islet cells. CONCLUSIONS: These results suggest that HGF and c-met may play a role in growth regulation in this organ.

New pancreas cancers cell lines that represent distinct stages of ductal differentiation.

BACKGROUND: Most available pancreas cancer cell lines have been in culture for long periods of time, have not been extensively characterized from the cell biology standpoint, or lack differentiated properties. EXPERIMENTAL DESIGN: We have established four new cell lines from ductal pancreatic cancers (IMIM-PC-1, IMIM-PC-2, SK-PC-1, and SK-PC-3). The phenotype and functional properties of the cell lines were analyzed using ultrastructural methods, antibodies detecting cytokeratin polypeptides and mucin epitopes, and cDNA probes of epithelial differentiation markers. RESULTS: IMIM-PC-2 and SK-PC-1 cells grow as a polarized monolayer, form domes, and express all CK polypeptides typical of simple epithelia and the MUC1 mucin. IMIM-PC-1 and SK-PC-3 are morphologically less differentiated and express low or undetectable levels of CK7 and MUC1. By Northern blotting, we found that SK-PC-1 and SK-PC-3 cells express carbonic anhydrase II and that the cystic fibrosis transmembrane regulator was undetectable in the four lines. Secretin induces a marked stimulation of cAMP levels in all cell lines except for SK-PC-3. Cytogenetic analysis demonstrates their human origin. CONCLUSIONS: Levels of CK7 and MUC1 are associated with less differentiated cultures. The new cell lines should be useful tools to study the cell biology of exocrine pancreas cancer.

Normal human pancreas cultures display functional ductal characteristics.

BACKGROUND: Normal cell cultures are invaluable in the analysis of cell differentiation and neoplastic transformation. EXPERIMENTAL DESIGN: We have developed methods to reproducibly culture normal pancreas epithelial cells. The characteristics of the cultures were analyzed using ultrastructural methods, antibodies, and cDNA probes detecting epithelial differentiation markers. RESULTS: Normal pancreas tissue (N = 56) was obtained from organ donors; isolation of highly enriched exocrine fraction consistently yielded epithelial cultures. In vitro proliferation assays revealed a lag growth phase of 2 days followed by a proliferative phase until 8. Epithelial cells formed a polarized monolayer displaying apical microvilli, tight junctions, and desmosomes. Zymogen granules were not observed. A panel of mouse monoclonal antibodies detecting differentiation antigens of epithelial cells was used to determine the phenotype of the cultures: all cells expressed cytokeratin polypeptides of simple epithelial (CK 7, CK 8, CK 18, and CK 19), whereas polypeptides typical of stratified epithelial (CK 5, CK 10, CK 13, and CK 16) were not detected. Cultured cells expressed the MUC1 apomucin as well as mucin-associated carbohydrate epitopes. Expression of the cystic fibrosis transmembrane regulator was demonstrated at the RNA level. Secretin induced a very high stimulation of cAMP levels. CONCLUSIONS: The ultrastructural characteristics, molecular markers, and hormone responsiveness of the cultures suggest a ductal cell phenotype. These cultures should be useful in the analysis of pancreas growth and differentiation.

Cytokeratins and mucins as molecular markers of cell differentiation and neoplastic transformation in the exocrine pancreas.

Acinar and ductal cells of the normal pancreas express cytokeratin (CK) patterns which indicate a heterogeneity of ductal epithelia. On account of the CK of pancreatic adenocarcinoma, it would seem to be probable that tumor epithelia possess a considerable potential for squamous epithelium metaplasia. Comparative studies of CK expression by ductal carcinoma of the pancreas and carcinoma of bile ducts, stomach and large intestine as well in cases of chronic pancreatitis have demonstrated the usefulness of CK for differential diagnosis of these conditions. From the number of apomucins, mainly MUC1 is produced in the normal pancreas. This capacity is maintained in pancreas carcinoma and cell lines derived from it.

Intermediate filaments as differentiation markers of exocrine pancreas. II. Expression of cytokeratins of complex and stratified epithelia in normal pancreas and in pancreas cancer.

Cytokeratin (CK) expression in tumors generally reflects the CK pattern of the corresponding normal epithelium. Pancreas cancers express CK of simple epithelia 7, 8, 18 and 19, as normal ductal cells. To analyze whether CK of complex or stratified epithelia are abnormally expressed in pancreas cancers, we have used polypeptide-specific mouse monoclonal antibodies (MAbs) detecting CK 5, CK 10, CK 13, CK 14 and CK 17, and an antibody detecting CK 13, CK 15 and CK 16. The streptavidin-peroxidase technique was applied on sections of fresh-frozen specimens of normal pancreas and of pancreas cancer. None of these polypeptides were expressed by normal acinar and centro-acinar cells. CK 5, CK 14 and CK 17 were expressed by less than 5% of cells in normal ducts, whereas CK 10, CK 13, CK 15 and CK 16 were not expressed at all. In tumors, CK 14, CK 15/16 and CK 17 were detected in the majority of cases studied; CK 5, CK 10 and CK 13 were present in a sub-population of pancreas cancers. CK of complex/stratified epithelia were detected in areas of glandular differentiation, but expression was more intense in areas of squamous differentiation. In pancreatitis adjacent to cancer, CK of complex/stratified epithelia were weakly detected or undetectable. These results suggest that up-regulation of these CK takes place in pancreas cancer. The CK phenotype may be of help in the differential diagnosis of this tumor.

Glycosyl phosphatidylinositol membrane anchoring of melanotransferrin (p97): apical compartmentalization in intestinal epithelial cells.

Melanotransferrin (p97) is an iron-binding membrane glycoprotein with 40% homology to transferrin and lactoferrin. It was first identified on the basis of its high level of expression in melanoma cells, as compared to normal melanocytes. It is also present in many cultured cell types. In normal tissues, p97 is expressed in fetal intestine, umbilical cord, sweat gland ducts and liver sinusoidal lining cells. Kinetic studies in melanoma cells have suggested that p97 plays a role in iron metabolism. We have examined expression of p97 in cell lines derived from human colorectal carcinomas which express a differentiated phenotype. When polarized, these cells showed a preferred apical distribution of p97, as demonstrated by immunohistochemistry, immune electron microscopy and domain-selective biotinylation. Correspondingly, p97 was only found on the apical brush border of epithelial cells in the fetal intestine. p97 was shown to be anchored to the membrane through a glycosyl phosphatidylinositol moiety by treatment with phophatidylinositol-specific phospholipase C (PI-PLC) and labeling with [14C]ethanolamine. These observations provide a basis for the elucidation of the physiological role of p97 in iron metabolism and its possible role in cell proliferation and malignant cell transformation.

Intestinal brush-border-associated enzymes: co-ordinated expression in colorectal cancer.

The brush border of normal small-intestine epithelial cells is rich in enzymes that are involved in the digestive process. Such molecules can be used as markers to analyze cell lineages and differentiation properties of colorectal cancers. Monoclonal antibodies detecting dipeptidyl peptidase-IV, aminopeptidase N, endopeptidase F, sucrase-isomaltase, alkaline phosphatase, maltase-glucoamylase and lactase have been used to analyze the phenotype of colorectal cancers, adjacent mucosa and histologically normal distant mucosa. The avidin-biotin peroxidase complex method was used. Expression of dipeptidyl peptidase-IV, aminopeptidase N, sucrase-isomaltase and alkaline phosphatase was common in non-neoplastic mucosa adjacent to, and distant from, the tumor; in contrast, endopeptidase F, maltase-glucoamylase and lactase were rarely expressed in normal distant mucosa and more frequently expressed in mucosa adjacent to the tumor. Dipeptidyl peptidase-IV, aminopeptidase N, endopeptidase F, sucrase-isomaltase and alkaline phosphatase were frequently expressed in colorectal cancers, whereas maltase-glucoamylase and lactase were rarely expressed. Two general patterns of antibody reactivity were observed: diffuse cytoplasmic and apical; apical reactivity was generally associated with more differentiated tumors. A logistic predictive regression model indicated that enzyme expression in colorectal cancers followed a coordinate pattern, but was unrelated to the location of the tumor, Dukes stage or differentiation grade. In conclusion, expression of brush-border-associated enzymes occurs frequently in colorectal cancers and is regulated in a co-ordinated manner. These markers can be used for the phenotypic sub-classification of colorectal cancers.