PubMed Health⌕ Search

Biomedical subjects

A G van Kessel

Publications and source records attributed to A G van Kessel.

At least 19 recordsLinked to original sources

Human mitochondrial complex I deficiency: investigating transcriptional responses by microarray.

NADH:ubiquinone oxidoreductase (complex I) deficiency is one of the most frequently encountered defects of the mitochondrial energy generating system. A deficiency of this enzyme complex leads to a wide variety in clinical disease expression. The cell biological consequences of such mutations, however, are poorly understood. We investigated transcriptional responses in fibroblast cell lines harboring mutations in the five different nuclear DNA encoded subunits using a mitochondria-targeting microarray. Expression profiles of cell lines cultured under conditions that favor glycolytic metabolism were compared to profiles when cultured under conditions favoring oxidative metabolism. Approximately 60 genes displayed differential expression under these conditions in either all mutated cell lines or selected cell lines only. A marked induction of metallothioneins as well as ATP1G1 transcripts was detected in all patient cell lines. Transcriptional responses such as the induction of heat shock protein transcripts, decreased PDK1,BNIP3 and mitochondrial genome encoding gene transcripts occurred in selected patient cell lines. The observed transcript profile points to a common, putative defensive, response relating to oxidative stress. Although further investigations of other human OXPHOS system diseases is warranted, these results clearly underline that functional genomics holds for the study of inherited metabolic disease.

Adult↗

Impairment of MAD2B-PRCC interaction in mitotic checkpoint defective t(X;1)-positive renal cell carcinomas.

The papillary renal cell carcinoma (RCC)-associated (X;1)(p11;q21) translocation fuses the genes PRCC and TFE3 and leads to cancer by an unknown molecular mechanism. We here demonstrate that the mitotic checkpoint protein MAD2B interacts with PRCC. The PRCCTFE3 fusion protein retains the MAD2B interaction domain, but this interaction is impaired. In addition, we show that two t(X;1)-positive RCC tumor cell lines are defective in their mitotic checkpoint. Transfection of PRCCTFE3, but not the reciprocal product TFE3PRCC, disrupts the mitotic checkpoint in human embryonic kidney cells. Our results suggest a dominant-negative effect of the PRCCTFE3 fusion gene leading to a mitotic checkpoint defect as an early event in papillary RCCs.

Amino Acid Sequence↗

Molecular mechanisms underlying human synovial sarcoma development.

Synovial sarcomas are rather common among soft-tissue tumors, occurring at any age but affecting mainly young adults. The vast majority of synovial sarcomas carries a t(X;18)(p11.2;q11.2) chromosomal translocation, in about one-third of the cases as the sole cytogenetic anomaly. Several studies have indicated that the t(X;18) translocation arises exclusively in synovial sarcomas, therefore being an excellent tool to diagnose this malignancy. The breakpoint-associated genes were recently isolated: SYT, from chromosome 18, and SSX1 and SSX2, both from the X chromosome. This discovery enabled the detection of SYT-SSX fusion transcripts by specific reverse transcriptase-polymerase chain reactions. This molecular genetics methodology has now been applied to numerous tumor samples and has led to the finding that, in contrast to tumors carrying SYT-SSX2 fusions, SYT-SSX1-positive tumors more often exhibit a biphasic histology, show a higher proliferation rate, and are associated with a poorer clinical outcome. It has also been shown that the SYT and SSX proteins are localized in the nucleus, where they appear to play a role in transcriptional regulation, SYT as an activator of transcription and the SSX proteins as transcriptional repressors. It was also found that SYT interacts and colocalizes in the nucleus with the BRM protein, a transcriptional coactivator, and that the SSX proteins colocalize in the nucleus with polycomb group proteins, which are transcriptional corepressors. Together, these studies have provided mechanistic clues about how the SYT-SSX fusion proteins may trigger synovial sarcoma development.

Amino Acid Sequence↗

Frequent loss of 9p21 (p16(INK4A)) and other genomic imbalances in human malignant fibrous histiocytoma.

To search for new recurrent genetic aberrations in malignant fibrous histiocytoma (MFH), a combination of conventional cytogenetic, comparative genomic hybridization (CGH), and Southern blot analyses was applied to a series of 34 tumors. Cytogenetic analysis revealed the presence of multiple structural and numerical aberrations, including marker chromosomes, telomeric associations, double minutes, and ring chromosomes. The most frequent genomic imbalances in this series of neoplasms as detected by CGH were gains of 1q21-q22 (69%), 17q23-qter (41%), and 20q (66%), and losses of 9p21-pter (55%), 10q (48%), 11q23-qter (55%), and 13q10-q31 (55%). Southern blot analyses with p16(INK4A) (CDKN2A; 9p21) and RB1 (13q14) probes provided clear indications for frequent deletions of these tumor suppressor genes, and as such, substantiated the CGH results. Additionally, examination of the TP53 and MDM2 genes showed frequent loss and amplification, respectively. These data indicate that genes involved in the RB1- and TP53-associated cell cycle regulatory pathways may play prominent roles in the development of human MFH.

Blotting, Southern↗

Control of O-glycan branch formation. Molecular cloning and characterization of a novel thymus-associated core 2 beta1, 6-n-acetylglucosaminyltransferase.

Core 2 O-glycan branching catalyzed by UDP-N-acetyl-alpha-D-glucosamine: acceptor beta1, 6-N-acetylglucosaminyltransferases (beta6GlcNAc-Ts) is an important step in mucin-type biosynthesis. Core 2 complex-type O-glycans are involved in selectin-mediated adhesion events, and O-glycan branching appears to be highly regulated. Two homologous beta6GlcNAc-Ts functioning in O-glycan branching have previously been characterized, and here we report a third homologous beta6GlcNAc-T designated C2GnT3. C2GnT3 was identified by BLAST analysis of human genome survey sequences. The catalytic activity of C2GnT3 was evaluated by in vitro analysis of a secreted form of the protein expressed in insect cells. The results revealed exclusive core 2 beta6GlcNAc-T activity. The product formed with core 1-para-nitrophenyl was confirmed by (1)H NMR to be core 2-para-nitrophenyl. In vivo analysis of the function of C2GnT3 by coexpression of leukosialin (CD43) and a full coding construct of C2GnT3 in Chinese hamster ovary cells confirmed the core 2 activity and failed to reveal I activity. The C2GnT3 gene was located to 5q12, and the coding region was contained in a single exon. Northern analysis revealed selectively high levels of a 5.5-kilobase C2GnT3 transcript in thymus with only low levels in other organs. The unique expression pattern of C2GnT3 suggests that this enzyme serves a specific function different from other members of the beta6GlcNAc-T gene family.

Amino Acid Sequence↗

Delineation of the protein domains responsible for SYT, SSX, and SYT-SSX nuclear localization.

In the vast majority of synovial sarcomas the N-terminal part of the SYT protein is fused to the C-terminal part of an SSX protein, either SSX1 or SSX2. The wild-type proteins, as well as the resultant SYT-SSX1 and SYT-SSX2 fusion proteins, are localized in the nucleus. Recent studies in experimental systems indicated that the SYT protein may function as a transcriptional activator whereas the SSX proteins may act as transcriptional repressors. In the present work we created a series of deletion mutants and found that SYT and SSX depend on N-terminal and highly conserved C-terminal domains for nuclear localization, respectively. Our results also show that the SYT-SSX proteins colocalize with SSX2, a feature that depends on the presence of the C-terminal SSX sequences in the chimeric proteins. Absence of these sequences led to an altered subcellular localization, coinciding with that of SYT. Besides, we found that endogenously expressed SSX proteins colocalize with polycomb-group proteins and condensed chromosomes during mitosis, features that are also conferred by the C-terminus of SSX. Taken together, these results led us to conclude that the SSX moiety, especially the most C-terminal 34 amino acids, of the SYT-SSX fusion proteins is crucial for aberrant spatial targeting and transcriptional control within the nucleus.

Amino Acid Sequence↗

Heterogeneous expression of the SSX cancer/testis antigens in human melanoma lesions and cell lines.

The SSX genes, located on the X chromosome, encode a family of highly homologous nuclear proteins. The SSX1 and SSX2 genes were initially identified as fusion partners of the SYT gene in t(X;18)-positive synovial sarcomas. Recently, however, it was found that these two genes, as well as the highly homologous SSX4 and SSX5 genes, are aberrantly expressed in different types of cancers, including melanomas. Because normal SSX expression has been detected only in the testis and, at very low levels, the thyroid, these proteins are considered as new members of the still growing family of cancer/testis antigens. These antigens are presently considered as targets for the development of cancer immunotherapy protocols. In the present study, we developed a monoclonal antibody found to recognize SSX2, SSX3, and SSX4 proteins expressed in formaldehyde-fixed and paraffin-embedded tissues. This antibody was used to investigate SSX expression in normal testis and thyroid, benign melanocytic lesions, melanoma lesions, and melanoma cell lines. SSX nuclear expression in the testis was found to be restricted to spermatogenic cells, mainly spermatogonia. Of 18 melanoma cell lines analyzed, 9 showed SSX RNA and protein expression, although heterogeneously and at variable levels. Treatment of an SSX-negative cell line with 5-aza-2'-deoxycytidine, a demethylating agent, led to SSX RNA and protein expression, indicating a role for methylation in transcription regulation. Thirty-four of 101 primary and metastatic melanoma cases and 2 of 24 common nevocellular and atypical nevus cases showed SSX nuclear staining. Again, SSX expression was heterogeneous, ranging from widespread to scarce. Our findings stress the importance of assessing the a priori SSX expression status of melanoma cases that may be selected for immunotherapeutic trials.

Adult↗

Nuclear localization and transactivating capacities of the papillary renal cell carcinoma-associated TFE3 and PRCC (fusion) proteins.

The papillary renal cell carcinoma-associated t(X;1)(p11;q21) leads to fusion of the transcription factor TFE3 gene on the X-chromosome to a novel gene, PRCC, on chromosome 1. As a result, two putative fusion proteins are formed: PRCCTFE3, which contains all known domains for DNA binding, dimerization, and transactivation of the TFE3 protein, and the reciprocal product TFE3PRCC. Upon transfection into COS cells, both wild type and fusion proteins were found to be located in the nucleus. When comparing the transactivating capacities of these (fusion) proteins, significant differences were noted. PRCCTFE3 acted as a threefold better transactivator than wild type TFE3 both in a TFE3-specific and in a general (Zebra) reporter assay. In addition, PRCC and the two fusion proteins were found to be potent transactivators in the Zebra reporter assay. We propose that, as a result of the (X;1) translocation, fusion of the N-terminal PRCC sequences to TFE3 alters the transactivation capacity of the transcription factor thus leading to aberrant gene regulation and, ultimately, tumor formation.

Animals↗

Cloning and characterization of a close homologue of human UDP-N-acetyl-alpha-D-galactosamine:Polypeptide N-acetylgalactosaminyltransferase-T3, designated GalNAc-T6. Evidence for genetic but not functional redundancy.

The UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T3, exhibits unique functions. Specific acceptor substrates are used by GalNAc-T3 and not by other GalNAc-transferases. The expression pattern of GalNAc-T3 is restricted, and loss of expression is a characteristic feature of poorly differentiated pancreatic tumors. In the present study, a sixth human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T6, with high similarity to GalNAc-T3, was characterized. GalNAc-T6 exhibited high sequence similarity to GalNAc-T3 throughout the coding region, in contrast to the limited similarity that exists between homologous glycosyltransferase genes, which is usually restricted to the putative catalytic domain. The genomic organizations of GALNT3 and GALNT6 are identical with the coding regions placed in 10 exons, but the genes are localized differently at 2q31 and 12q13, respectively. Acceptor substrate specificities of GalNAc-T3 and -T6 were similar and different from other GalNAc-transferases. Northern analysis revealed distinct expression patterns, which were confirmed by immunocytology using monoclonal antibodies. In contrast to GalNAc-T3, GalNAc-T6 was expressed in WI38 fibroblast cells, indicating that GalNAc-T6 represents a candidate for synthesis of oncofetal fibronectin. The results demonstrate the existence of genetic redundancy of a polypeptide GalNAc-transferase that does not provide full functional redundancy.

Amino Acid Sequence↗

Control of O-glycan branch formation. Molecular cloning of human cDNA encoding a novel beta1,6-N-acetylglucosaminyltransferase forming core 2 and core 4.

A novel human UDP-GlcNAc:Gal/GlcNAcbeta1-3GalNAcalpha beta1, 6GlcNAc-transferase, designated C2/4GnT, was identified by BLAST analysis of expressed sequence tags. The sequence of C2/4GnT encoded a putative type II transmembrane protein with significant sequence similarity to human C2GnT and IGnT. Expression of the secreted form of C2/4GnT in insect cells showed that the gene product had UDP-N-acetyl-alpha-D-glucosamine:acceptor beta1, 6-N-acetylglucosaminyltransferase (beta1,6GlcNAc-transferase) activity. Analysis of substrate specificity revealed that the enzyme catalyzed O-glycan branch formation of the core 2 and core 4 type. NMR analyses of the product formed with core 3-para-nitrophenyl confirmed the product core 4-para-nitrophenyl. The coding region of C2/4GnT was contained in a single exon and located to chromosome 15q21.3. Northern analysis revealed a restricted expression pattern of C2/4GnT mainly in colon, kidney, pancreas, and small intestine. No expression of C2/4GnT was detected in brain, heart, liver, ovary, placenta, spleen, thymus, and peripheral blood leukocytes. The expression of core 2 O-glycans has been correlated with cell differentiation processes and cancer. The results confirm the predicted existence of a beta1,6GlcNAc-transferase that functions in both core 2 and core 4 O-glycan branch formation. The redundancy in beta1,6GlcNAc-transferases capable of forming core 2 O-glycans is important for understanding the mechanisms leading to specific changes in core 2 branching during cell development and malignant transformation.

Amino Acid Sequence↗

A novel chromosomal region of allelic loss, 4q32-q34, in human osteosarcomas revealed by representational difference analysis.

Representational difference analysis (RDA) of a human osteosarcoma xenograft resulted in the isolation of four tumor-associated homozygously deleted DNA fragments, all originating from chromosome 4, region q32-q34. Southern blot analysis using the RDA fragments and interphase FISH analysis using PACs corresponding to these RDA fragments revealed allelic loss of the 4q32-q34 region in 17 of 27 (63%) osteosarcomas tested. These results suggest the involvement of tumor suppressor gene(s) within this chromosomal region in osteosarcoma development. The RDA fragments and corresponding PAC clones will be instrumental in the isolation of such gene(s). Genes Chromosomes Cancer 26:115-124, 1999.

Animals↗

Cloning of a human UDP-N-acetyl-alpha-D-Galactosamine:polypeptide N-acetylgalactosaminyltransferase that complements other GalNAc-transferases in complete O-glycosylation of the MUC1 tandem repeat.

A fourth human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T4, was cloned and expressed. The genomic organization of GalNAc-T4 is distinct from GalNAc-T1, -T2, and -T3, which contain multiple coding exons, in that the coding region is contained in a single exon. GalNAc-T4 was placed at human chromosome 12q21.3-q22 by in situ hybridization and linkage analysis. GalNAc-T4 expressed in Sf9 cells or in a stably transfected Chinese hamster ovary cell line exhibited a unique acceptor substrate specificity. GalNAc-T4 transferred GalNAc to two sites in the MUC1 tandem repeat sequence (Ser in GVTSA and Thr in PDTR) using a 24-mer glycopeptide with GalNAc residues attached at sites utilized by GalNAc-T1, -T2, and -T3 (TAPPAHGVTSAPDTRPAPGSTAPPA, GalNAc attachment sites underlined). Furthermore, GalNAc-T4 showed the best kinetic properties with an O-glycosylation site in the P-selectin glycoprotein ligand-1 molecule. Northern analysis of human organs revealed a wide expression pattern. Immunohistology with a monoclonal antibody showed the expected Golgi-like localization in salivary glands. A single base polymorphism, G1516A (Val to Ile), was identified (allele frequency 34%). The function of GalNAc-T4 complements other GalNAc-transferases in O-glycosylation of MUC1 showing that glycosylation of MUC1 is a highly ordered process and changes in the repertoire or topology of GalNAc-transferases will result in altered pattern of O-glycan attachments.

Amino Acid Sequence↗

Sublocalization of the breakpoints of a t(5;16) in myelodysplasia.

As a first step in characterizing a t(5;16)(q31;p11.2) in a patient with the diagnosis refractory anemia with ring sideroblasts, a cell fusion was carried out between bone marrow cells from the patient and the Chinese hamster cell line A3. Using PCR and FISH analysis on hybrid lines containing the human derivative 16 chromosome, the breakpoints could be mapped between the markers TCF-7 and IL-9 on chromosome 5 and OL-7 and s30A4 on chromosome 16, both regions spanning approximately 1 Mb. Since the breakpoint on 5q has occurred in a region that is frequently deleted in myeloid malignancies, the gene disrupted by this translocation could also be implicated in this aberration.

Animals↗

Identification of the critical region of 12p over-representation in testicular germ cell tumors of adolescents and adults.

Cytogenetically, testicular germ cell tumors of adolescents and adults (TGCTs) are characterized by gain of 12p-sequences, most often through isochromosome formation (i(12p)). Fluorescence in situ hybridization (FISH) has shown that i(12p))-negative TGCTs also cryptically contain extra 12p-sequences. The consistency of 12p-over-representation in all histological subtypes of TGCTs, including their preinvasive stage, suggests that gain of one or more genes on 12p is crucial in the development of this cancer. So far, studies aimed at the identification of the relevant gene(s) were based on the 'candidate-gene approach'. No convincing evidence in favor of or against a particular gene has been reported. We combined conventional karyotyping, comparative genomic hybridization, and FISH to identify TGCTs with amplifications of restricted regions of 12p. Out of 49 primary TGCTs (23 without i(12p), 13 with and 13 unknown), eight tumors (six without i(12p) and two unknown) showed amplifications corresponding to 12p11.1-p12.1. Using bicolour-FISH, physical mapping, and semi-quantitative polymerase chain reactions, the size of the shortest region of overlap of amplification (SROA) was estimated to be between 1750-3000 kb. In addition, we mapped a number of genes in and around this region. While fourteen known genes could be excluded as candidates based on their location outside this region, we demonstrate that KRAS2, JAW1 and SOX5 genes are localized within the SROA. While KRAS2 and JAW1 map to the proximal border of the SROA, SOX5 maps centrally in the SROA. KRAS2 and JAW1 are expressed in all TGCTs, whereas one 12p amplicon-positive TGCT lacks expression of SOX5. The critical region of 12p over-represented in TGCTs is less than 8% of the total length of the short arm of chromosome 12. It will be helpful in the identification of the gene(s) involved in TGCT-development.

Adolescent↗

Genomic organization and chromosomal localization of three members of the UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyltransferase family.

A homologous family of UDP- N -acetylgalactosamine: polypeptide N -acetylgalactosaminyltransferases (GalNAc-transferases) initiate O-glycosylation. These transferases share overall amino acid sequence similarities of approximately 45-50%, but segments with higher similarities of approximately 80% are found in the putative catalytic domain. Here we have characterized the genomic organization of the coding regions of three GalNAc-transferase genes and determined their chromosomal localization. The coding regions of GALNT1 , -T2 , and -T3 were found to span 11, 16, and 10 exons, respectively. Several intron/exon boundaries were conserved within the three genes. One conserved boundary was shared in a homologous C. elegans GalNAc-transferase gene. Fluorescence in situ hybridization showed that GALNT1 , -T2 , and -T3 are localized at chromosomes 18q12-q21, 1q41-q42, and 2q24-q31, respectively. These results suggest that the members of the polypeptide GalNAc-transferase family diverged early in evolution from a common ancestral gene through gene duplication.

Amino Acid Sequence↗

Assignment of Ptprn2, the gene encoding receptor-type protein tyrosine phosphatase IA-2beta, a major autoantigen in insulin-dependent diabetes mellitus, to mouse chromosome region 12F.

The receptor-type protein tyrosine phosphatase IA-2beta gene (mouse gene symbol Ptprn2) encodes a major autoantigen in insulin-dependent diabetes mellitus. We physically mapped Ptprn2 by fluorescence in situ hybridization to band F of mouse chromosome 12, a region that lacks diabetes susceptibility loci. The mapping confirms the proposed synteny of mouse 12F with band q36 of human chromosome 7.

Animals↗