bromochloroacetic-acid and Chromosome-Deletion

Differential diagnosis of monophasic synovial sarcoma requires the detection of specific biological markers. In this study we evaluated the presence of molecular alterations in 15 monophasic synovial sarcomas. Multiple changes affecting chromosome arms were detected by CGH-array in all microdissected cases available, and an association between gain or loss of specific regions harbouring cancer progression-associated genes and aneuploid status was found. The most frequent alteration was loss of 3p including 3p21.3-p23 region that, however, did not involve the promoter regions of the corresponding genes, RASSF1 and MLH1. Using Real-Time PCR, mRNA levels of both resulted moderately high compared to normal tissue; however, the weak to absent protein expression suggests RASSF1 and MLH1 post-transcription deregulation. Moreover, immunohistochemical analysis revealed that both mesenchymal and epithelial antigens were present in diploid tumours. These findings confirm the genetic complexity of monophasic synovial sarcoma and underline the need to integrate different analyses for a better knowledge of this tumour, essential to investigate new diagnostic and prognostic markers.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Biomarkers, Tumor; Carrier Proteins; Chromosome Deletion; Chromosomes, Human, Pair 3; DNA, Neoplasm; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Keratins; Male; Microsatellite Repeats; Middle Aged; Mucin-1; MutL Protein Homolog 1; Neoplasms, Connective Tissue; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Prognosis; RNA, Messenger; Sarcoma, Synovial; Transcription, Genetic; Tumor Suppressor Proteins; Vimentin

A candidate tumor suppressor gene, WT-1, is believed to have an important role in the pathogenesis of Wilms' tumor, especially that occurring in patients with congenital aniridia.. To obtain a stable tumor line to work with, Wilms' tumor tissue was serially transplanted in athymic nude mice. Biopsied Wilms' tumor tissue, derived from an aniridia patient, was transplanted subcutaneously to an athymic nude mouse, and then transplanted serially. Histopathologic and molecular biologic studies were performed on the xenotransplants.. The aniridia patient showed partial deletion in one short arm of chromosome 11, which bears the WT-1 gene. The tumor was successfully transplanted in the nude mouse. Although the tumor contained blastemic, organoid, and stromal histologic elements, the organoid element began to decrease after more than 20 passages. Cytogenetic analysis revealed an additional abbreviation of one long arm of chromosome 6. Dot blot analysis showed that the copy number of WT-1 gene was decreased to half the amount in the tumor, in spite of the WT-1 transcript with normal size detected by Northern blotting.. The tumor is expected to bear one WT-1 gene with minute abnormalities as well as one congenitally deleted gene. This tumor line is useful when examining the effect caused by introduction of WT-1 gene to Wilms' tumor in vivo.

Topics: Alleles; Animals; Aniridia; Biopsy; Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 11; Genes, Wilms Tumor; Humans; Immunohistochemistry; Infant; Keratins; Kidney Neoplasms; Male; Mice; Mice, Nude; Microscopy, Electron; Mucin-1; Mucoproteins; Neoplasm Proteins; S100 Proteins; Transplantation, Heterologous; Uromodulin; Vimentin; Wilms Tumor

Bellini duct carcinoma (BDC) is a rare and highly aggressive renal tumor whose histogenesis is still a matter of debate although a putative origin from collecting ducts has been proposed.. A primary tumor cell culture was obtained from a BDC of a 57-year-old man who presented with a mass of the right kidney. The patient died from disease progression 18 months after diagnosis. The light and ultrastructural features were consistent with previous reports on BDC. The expression of low (Ker 18) and high (Ker 5, Ker 8, Ker 10) molecular weight keratins was studied.. The BDC tumor cells displayed strong positivity for keratins, 5, 8 and 18 but did not react with the anti-keratin 10 antibody. Northern blot analysis of total mRNA revealed expression of the c-erbB-1 oncogene unlike two conventional clear cell carcinomas of the kidney used as control. Cytogenetic analysis revealed an aneuploid karyotype: 53,XY,del(1)(p34),+iso(1q),+iso(5p),+4,+7,+8,-14,del(16)(q22). No submicroscopic deletion on p14-21 and p26 regions of the short arm of chromosome 3 was detected on Southern blot analysis.. The absence of structural changes in the short arm of chromosome 3 (usually present in hereditary and sporadic renal cell carcinomas) in the presence of chromosomal abnormalities observed in malignant lesions of urothelial origin confers to BDC a unique genetic profile among papillary tumors of the kidney.

Topics: Carcinoma, Papillary; Chromosome Deletion; Chromosomes, Human, Pair 1; Chromosomes, Human, Pair 3; Chromosomes, Human, Pair 7; ErbB Receptors; Humans; Karyotyping; Keratins; Kidney Neoplasms; Kidney Tubules, Collecting; Male; Middle Aged; Monosomy; Trisomy

A human hepatocyte line (HHY41) was established from normal human liver tissue. This cell line was derived from a primary culture of human hepatocytes maintained between two layers of collagen gel for 4 weeks. It differs from other human hepatocyte lines in that transfection with the simian virus 40 gene was not used for cellular transformation and nonhepatocellular coculture cells were not present. HHY41 cells have proliferated freely in serum and hormone-supplemented medium after more than 1 year in continuous culture, exhibiting typical morphological characteristics of hepatocytes. HHY41 cells retain glucose-6-phosphatase activity. They also retain the ability to secrete liver-specific proteins such as albumin, transferrin, and alpha-fetoprotein. Northern blot analysis confirmed the presence of albumin mRNA. Cytochromes P450 induced by polycyclic aromatic hydrocarbons are maintained in these cells. Detection of cell surface antigens revealed that HHY41 cells express alpha 1 beta 1-integrin, which is expressed by normal hepatocytes and not by bile duct epithelial cells. High-molecular-weight cytokeratin, a marker for bile duct cells, is also absent in HHY41. Cytogenetic analysis showed hyperdiploid karyotype with a consistent deletion in the short arm of chromosome 1. HHY41 can be considered a new human hepatocyte line which retains liver-specific functions of differentiated hepatocytes. Derived from normal liver tissue, not a hepatocellular carcinoma, it provides a new model system for studying the regulation of cell growth and differentiated functions in human hepatocytes.

Topics: Adult; Aneuploidy; Cell Division; Cell Line; Chromosome Deletion; Chromosomes, Human, Pair 1; Collagen; Gels; Glucose-6-Phosphatase; Humans; Integrin alpha1beta1; Integrins; Karyotyping; Keratins; Liver; Male; Proteins; RNA, Messenger

Among the more than 30 different human proteins of the cytokeratin (CK) group of intermediate filament (IF) proteins, the significance of the epidermal polypeptide CK 2 (Moll et al., 1982, Cell 31, 11-24) has been repeatedly questioned in the literature. Here, we show, by in vitro translation and protein gel electrophoresis, that human epidermis from various body sites does indeed contain relatively large amounts of mRNA encoding a distinct polypeptide comigrating with native epidermal CK 2. We also report the isolation of a cDNA clone encoding the complete sequence of CK 2, which is a type II CK different from--but related to--epidermal CKs 1 and 5 on the one hand and corneal CK 3 on the other. The mRNA of approximately 2.6 kb encodes a polypeptide of 645 amino acids and M(r) 65,852, in good agreement with the value of 65.5 kDa previously estimated from gel electrophoresis. This human CK, the largest so far known, displays several features typical of CKs of stratified epithelia, including numerous repeats of glycine-rich tetrapeptides in the head and tail domains. Northern blot and in situ hybridizations have shown that CK 2 is expressed strictly suprabasally, usually starting in the third or fourth cell layer of epidermis, and this was confirmed at the protein level by immunohistochemistry using CK 2-specific antibodies. The protein has been detected as a regular epidermal component in skin samples from different body sites, albeit as a minor CK in "soft skin" (e.g., breast nipple, penile shaft, axilla), but not in foreskin epithelium and in other epithelia, in squamous metaplasias and carcinomas, or in cultured cell lines derived therefrom. We propose that CK 2 is a late cytoskeletal IF addition synthesized during maturation of epidermal keratinocytes which probably contributes to terminal cornification.

Topics: Amino Acid Sequence; Animals; Antibodies; Base Sequence; Blotting, Southern; Cell Differentiation; Chromosome Deletion; Cloning, Molecular; Cytoskeleton; DNA; Epidermal Cells; Epidermis; Female; Humans; Keratins; Male; Molecular Sequence Data; Mouth Mucosa; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Protein Biosynthesis; Protein Conformation; Rabbits; Reticulocytes; RNA; Sequence Homology, Nucleic Acid; Skin

To elucidate the elements required for regulation of keratin expression in epidermis, we have linked a short, 300 base pair segment, corresponding to the promoter region of a human K#14 gene, to the chloramphenicol-acetyl-transferase gene. This construct was introduced into various mammalian cell lines and primary cultures via Ca3(PO4)2 precipitation. The 300 base pair segment from the keratin gene promoter region was active in all epithelial cells studied including transformed, simple epithelial cells such as HeLa and ME-180, cell lines derived from stratified epithelium, such as SCC-12, as well as primary cultures of epithelial cells. The construct was inactive in all non-epithelial cells tested including fibroblasts and melanocytes. The segment does not function as a silencer in nonepithelial cells but it can function as an enhancer in epithelial cells. Using the polymerase chain reaction we have constructed a series of deletions of the promoter and have localized an essential function within a 40 bp sequence. We conclude that we have identified the keratin gene promoter that is sufficient to confer epithelial-specific expression.

Topics: Animals; Base Composition; Base Sequence; Cell Line; Chloramphenicol O-Acetyltransferase; Chromosome Deletion; Epithelium; Gene Expression; Genes; Humans; Keratins; Molecular Sequence Data; Oligonucleotide Probes; Organ Specificity; Promoter Regions, Genetic; Pseudogenes; Transfection; Tumor Cells, Cultured

To study the role of the amino-terminal domain of the desmin subunit in intermediate filament (IF) formation, several deletions in the sequence encoding this domain were made. The deleted hamster desmin genes were fused to the RSV promoter. Expression of such constructs in vimentin-free MCF-7 cells as well as in vimentin-containing HeLa cells, resulted in the synthesis of mutant proteins of the expected size. Single- and double-label immunofluorescence assays of transfected cells showed that in the absence of vimentin, desmin subunits missing amino acids 4-13 are still capable of filament formation, although in addition to filaments large numbers of desmin dots are present. Mutant desmin subunits missing larger portions of their amino terminus cannot form filaments on their own. It may be concluded that the amino-terminal region comprising amino acids 7-17 contains residues indispensable for desmin filament formation in vivo. Furthermore it was shown that the endogenous vimentin IF network in HeLa cells masks the effects of mutant desmin on IF assembly. Intact and mutant desmin colocalized completely with endogenous vimentin in HeLa cells. Surprisingly, in these cells endogenous keratin also seemed to colocalize with endogenous vimentin, even if the endogenous vimentin filaments were disturbed after expression of some of the mutant desmin proteins. In MCF-7 cells some overlap between endogenous keratin and intact exogenous desmin filaments was also observed, but mutant desmin proteins did not affect the keratin IF structures. In the absence of vimentin networks (MCF-7 cells), the initiation of desmin filament formation seems to start on the preexisting keratin filaments. However, in the presence of vimentin (HeLa cells) a gradual integration of desmin in the preexisting vimentin filaments apparently takes place.

Topics: Amino Acid Sequence; Animals; Biopolymers; Cell Line; Chromosome Deletion; Cricetinae; Desmin; Fluorescent Antibody Technique; Humans; Intermediate Filaments; Keratins; Microscopy, Fluorescence; Molecular Sequence Data; Mutation; Nuclear Envelope; Structure-Activity Relationship; Transfection; Vimentin

To investigate the sequences important for assembly of keratins into 10-nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.

Topics: Amino Acid Sequence; Cells, Cultured; Chromosome Deletion; Epidermis; Escherichia coli; Humans; Intermediate Filaments; Keratins; Macromolecular Substances; Molecular Sequence Data; Mutagenesis; Peptide Fragments; Phenotype; Recombinant Proteins; Structure-Activity Relationship; Transfection

The keratinocyte is a major cell type of the body, and in epidermis, keratinocytes have potential for future gene targeting and drug therapy. Despite the importance of keratinocytes in cell biology and medicine, little is known about the molecular mechanisms underlying keratinocyte-specific gene expression. Here, we report the first detailed characterization of the sequences and factors controlling expression of a human gene expressed specifically in keratinocytes. Using 5' upstream sequence of the human K14 keratin gene coupled to one of two reporter genes, we examined sequences necessary and sufficient for expression of K14 in both cultured human keratinocytes and in mitotically active basal keratinocytes of transgenic mouse epidermis. We demonstrated the existence of distal and proximal elements located 5' from the transcription initiation site of the hK14 gene, which when combined with a TATA box element, appear to act in concert to drive keratinocyte-specific expression. We examined the proximal region in detail. After using CAT assays to narrow a transcriptional activation element to within 110 bp, we demonstrated the existence of a keratinocyte nuclear factor which binds to a 10-bp palindrome, 5'-GCCTGCAGGC-3', within this domain. Using methylation interference analysis, we identified the G residues important for factor binding, and showed that point mutations in these G residues not only blocked factor binding but also resulted in decreased transcriptional activity of an hK14-CAT gene. The factor was most abundant in keratinocytes, was expressed at lower levels in some simple epithelial cell lines, and was not detected in fibroblasts or lymphoma cells. Moreover, the 10-bp sequence was similar to sequences found in the 5' upstream sequences of several other genes expressed in keratinocytes, and at least one of these genes, the human K1 gene, contained a sequence that competed with the hK14 proximal element for binding factor. Collectively, our data suggest that both the sequence and the nuclear factor that we have identified may be involved in controlling keratinocyte-specific expression in vitro and in vivo.

Topics: Animals; Base Sequence; Binding Sites; Cell Nucleus; Chromosome Deletion; Cloning, Molecular; Epidermis; Gene Expression Regulation; Genes; Humans; Introns; Keratinocytes; Keratins; Kinetics; Mice; Mice, Transgenic; Molecular Sequence Data; Mutagenesis; Nuclear Proteins; Plasmids; Restriction Mapping; Transcription, Genetic; Transfection

We have deleted cDNA sequences encoding portions of the amino- and carboxy-terminal end of a human type I epidermal keratin K14, and examined the molecular consequences of forcing the expression of these mutants in simple epithelial and squamous cell carcinoma lines. To follow the expression of our mutant products in transfected cells, we have tagged the 3' end of the K14 coding sequence with a sequence encoding an antigenic domain of the neuropeptide substance P. Using DNA transfection and immunohistochemistry (with an antibody against substance P), we have defined the limits of K14 sequence necessary to incorporate into a keratin filament network in vivo without disrupting its architecture. We have also uncovered major differences in the behavior of carboxy- and amino-terminal alpha-helical mutants which do perturb the cytoskeletal network of IFs: whereas carboxy terminal mutants give rise to aggregates of keratin in the cytoplasm, amino-terminal mutants tend to produce aggregates of keratins which seem to localize at the nuclear surface. An examination of the phenotypes generated by the carboxy and amino-terminal mutants and the behavior of cells at late times after transfection suggests a model whereby initiation of filament assembly occurs at discrete sites on the nuclear envelope and filaments grow from the nucleus toward the cytoplasm.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Line; Chromosome Deletion; Codon; Cytoskeleton; DNA; Epithelium; Fluorescent Antibody Technique; Genes; Intermediate Filaments; Keratins; Molecular Sequence Data; Mutation; Plasmids; Transfection

XK81A1 is a type I epidermal keratin gene expressed in early developmental stages of Xenopus (Jonas et al. 1985). Fusion of the keratin promoter (-5900 to +26) to a human beta globin gene led to fully epidermis-specific accumulation of human globin mRNA and protein when this DNA was injected into fertilized eggs. Further localization of regulatory sequences was accomplished by injecting marked, 5'-deleted keratin gene DNA into fertilized eggs and evaluating tissue specificity of expression. All 5' flanking DNA upstream from -487 could be removed without interfering with keratin gene expression or regulation. These results suggest that the primary mode of regulation of epidermis-specific keratin gene expression is at the level of transcription, and that sequence elements in the 5' flanking region of the keratin gene, between -487 and +26, are responsible for this regulation.

Topics: Animals; Base Sequence; Chromosome Deletion; Chromosome Mapping; DNA, Recombinant; Epidermis; Female; Gene Expression Regulation; Globins; Keratins; Molecular Sequence Data; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Transcription, Genetic; Transfection; Xenopus laevis

We have deleted cDNA sequences encoding portions of the carboxy-terminal end of a human type I epidermal keratin K14, and examined the molecular consequences of forcing the expression of these mutants in simple epithelial and squamous cell carcinoma lines. To follow the expression of our mutant products in transfected cells, we have tagged the 3' end of the K14 coding sequence with a sequence encoding an antigenic domain of the neuropeptide substance P. Using DNA transfection and immunohistochemistry (with an antibody against substance P), we have identified a collection of mutants that have a wide range of morphological effects on the endogenous keratin filament networks of transfected cells. Mutants that are missing most of the nonhelical carboxy-terminal domain of K14 incorporate into the endogenous keratin filaments without any visible perturbations on the network. In contrast, mutants that are missing as few as 10 of the 310 amino acids of the central alpha-helical domain of the polypeptide cause gross alterations in the keratin network. In some cases, the entire cytoskeletal network of keratins was disrupted, leaving no evidence of 8-nm filaments. These results reveal the existence of a dynamic exchange between newly synthesized subunits and preexisting keratin filaments.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carcinoma, Squamous Cell; Cell Line; Chromosome Deletion; DNA; Epithelium; Fluorescent Antibody Technique; Humans; Keratins; Mutation; Plasmids; Skin; Transcription, Genetic; Transformation, Genetic

A cell line (T3/73) from a Wilms' tumour has been established from a 9 month-old boy with aniridia. The tumour was removed in 1973. On histological examination a diagnosis of Wilms' tumour was made which showed undifferentiated areas, marked tubule formation and abundant striped muscle fibres. The tumour cells, which are fusiform, grew rapidly in culture without the addition of growth factors, and have undergone over 100 passages. Approximately 95% and 5% were positive for desmin and cytokeratin, respectively. The cell doubling time was 28 hr. Cytogenetic studies revealed a karyotype of 46,XY,del(11) (p12::p14). Although the cells stained very intensely with a monoclonal antibody that detects oncogene ras p 21 antigen, Southern blot analysis using c-Ha-ras as a probe failed to reveal an obvious deletion or amplification of either Ha-ras allele.

Topics: Antibodies, Monoclonal; Cell Line; Chromosome Deletion; Chromosomes, Human, Pair 11; Desmin; Gene Amplification; Humans; Infant; Karyotyping; Keratins; Male; Oncogenes; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Wilms Tumor

Using recombination of an appropriate expression vector system (pINDU) with a complete cDNA encoding a basic (type II) cytokeratin, i.e. cytokeratin 8 (1) of Xenopus laevis, we transformed Escherichia coli cells to synthesize considerable amounts of an insoluble eukaryotic cytoskeletal protein. The cytokeratin was deposited in large 'inclusion bodies' in the bacterial cytoplasm but did not form detectable filamentous structures. However, when the E. coli-expressed cytokeratin was purified and combined in vitro with an authentic cytokeratin of the complementary, i.e. acidic (type I) subfamily, it formed typical intermediate-sized filaments (IFs). Using Bal31 deletion from either the 5' or the 3' end of the cDNA, series of polypeptides progressively deleted from the amino or the carboxy terminus were produced in E. coli and identified by monoclonal antibodies. These assays allowed the mapping of epitopes. The deletion polypeptides of cytokeratin 8 were further examined to localize the region(s) involved in the heterotypic binding of alpha-helices of type I cytokeratins, using an in vitro nitrocellulose blot binding assay. We show that a region of 37 amino acids located in the central portion of coil 2 of the alpha-helical rod domain is sufficient for the specific recognition of a radiolabelled type I cytokeratin, i.e. cytokeratin 18 (D) from rat liver. In addition, deletion polypeptides containing only coil 1 of the alpha-helical rod also bind strongly the complementary cytokeratin. This indicates that the capability of heterotypic recognition and complex formation is not restricted to a single signal sequence but is located in distant and independent alpha-helical domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Animals; Chromosome Deletion; Cloning, Molecular; DNA; Escherichia coli; Genes; Genetic Vectors; Keratins; Molecular Sequence Data; Protein Biosynthesis; Recombinant Proteins; Transcription, Genetic; Xenopus laevis