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K C Arden

Publications and source records attributed to K C Arden.

At least 19 recordsLinked to original sources
PubMed1998

Elevated cyclins and cyclin-dependent kinase activity in the rhabdomyosarcoma cell line RD.

An important early event in the differentiation of skeletal muscle cells is exit from the cell cycle, after which full expression of the muscle phenotype occurs. Rhabdomyosarcoma (RMS), a tumor of skeletal muscle origin, expresses a number of muscle-specific proteins, including MyoD; however, these cells fail to arrest or differentiate when cultured in differentiation medium (DM). To determine the basis for the failure of RMS cells to differentiate or arrest, we studied the molecular response of the embryonal RMS cell line, RD, to culture in DM. Under these conditions, the retinoblastoma protein (RB) was primarily in the hyperphosphorylated state. This is in contrast to myoblasts cultured in DM, in which the hypophosphorylated form of RB is exclusively present. Measurements of the expression and activities of cyclin-dependent kinases (cdks) cdk2 and cdk4 indicated that RD cells maintained higher levels than do myoblasts, and the activity and abundance of these proteins did not significantly decrease upon culture in DM in RD cells, as they did in myoblasts. Similarly, elevated expression of cyclins D1, E, and A was observed in RD cells. Interestingly, cdk inhibitors are expressed in RD cells, with p16ink4 expression markedly elevated relative to myoblasts. Ectopic expression of p21cip1, p16ink4, or p27kip1 caused a growth arrest of RD cells but not detectable expression of a myogenic marker. Furthermore, a constitutively active RB protein could also inhibit the growth of RD cells without inducing myogenic differentiation. Taken together, these data suggest that the elevated levels of cdk2 and/or cdk4 observed in RD cells contribute to the inability of RD cells to growth arrest when cultured in DM but that these activities alone are not responsible for the failure of RD cells to differentiate.

Cell Cycle↗
PubMed1998

Identification of a new MAGE gene with tumor-specific expression by representational difference analysis.

Human genes expressed exclusively in tumors and male germ line cells, such as those of the MAGE, BAGE, and GAGE families, encode antigens recognized by T lymphocytes, which are potentially useful for antitumor immunotherapy. To identify new genes of this type, we generated cDNA populations enriched in sequences expressed only in testis and melanoma, using the representational difference analysis approach. A testis cDNA library enriched by subtraction with cDNA from four other normal tissues was hybridized with radiolabeled melanoma cDNA enriched by subtraction with normal skin cDNA. A cDNA fragment sharing significant homology with MAGE genes was identified, and a cosmid containing this new gene, named MAGE-C1, was isolated. MAGE-C1 is composed of four exons and encodes a putative protein of 1142 amino acids. It is about 800 residues longer than the other MAGE proteins due to the insertion of a large number of short repetitive sequences in front of the MAGE-homologous sequence. The MAGE-C1 gene appears to be located on band Xq26, whereas the MAGE-A and MAGE-B genes are located on Xq28 and Xp21, respectively. Like other MAGE genes, MAGE-C1 is expressed in a significant proportion of tumors of various histological types, whereas it is silent in normal tissues except testis. It is probable, therefore, that like other MAGE genes, MAGE-C1 encodes antigens that may constitute useful targets for cancer immunotherapy because of their strict tumoral specificity.

Amino Acid Sequence↗
PubMed1998

Genetic instability leads to loss of both p53 alleles in a human glioblastoma.

Little is known about the relationship between genetic recombination mechanisms and loss of tumour suppressor genes in solid tumours. Here, we demonstrate deletion and truncation of both p53 alleles in a primary human glioblastoma and a derived cell line as the combined result of a t(17;20) reciprocal translocation and a 1.1 Mbp genomic deletion on chromosome 17p, starting in intron 4 of the p53 gene and ending at the telomeric CA-repeat marker D17S960. These results (i) suggest that genetic instability can lead to loss of tumour suppressor gene function in solid cancers, (ii) provide mapping of one such recombination event at the nucleotide level, and (iii) establish the orientation of the p53 gene on chromosome 17 as: centromere 5'-3'-telomere.

Alleles↗
PubMed1998

Cloning and characterization of three human forkhead genes that comprise an FKHR-like gene subfamily.

Alveolar rhabdomyosarcomas are associated with unique chromosomal translocations t(2;13) and t(1;13), which arise from fusion of the genes for the paired box proteins PAX3 and PAX7, respectively, to the FKHR (forkhead in rhabdomyosarcoma) gene on chromosome 13q14. Here we report the identification and characterization of three novel human forkhead genes with similarity to FKHR. The three genes (HGMW-approved symbols FKHRP1, FKHRL1, and FKHRL1P1) map to chromosomal regions 5q35.2-q35.3, 6q21, and 17p11, respectively. Based on amino acid sequence comparisons of their forkhead domains, FKHRL1, FKHRL1P1, and FKHRP1 share 86, 84, and 68% identity, respectively, with FKHR. While FKHR and FKHRL1 are expressed in every human adult tissue examined, FKHRP1 mRNA expression could not be detected, and FKHRL1P1 expression was present only at low levels. FKHR and FKHRL1 share a similar genomic organization, each having a very large intron 1 (FKHR approximately 130 kb and FKHRL1 > 90 kb), which bisects their respective forkhead domains at identical positions, as well as a second intron just downstream of each stop codon. FKHRP1 and FKHRL1P1 lack introns and contain stop codons that prevent them from yielding full-length proteins. Thus, while FKHR and FKHRL1 represent functional genes, FKHRP1 and FKHRL1P1 probably are processed pseudogenes. These results suggest that these four genes represent an FKHR-like gene subfamily within the larger human forkhead gene family.

Amino Acid Sequence↗
PubMed1997

Chromosomal mapping and mutational analysis of the coding region of the glycogen synthase kinase-3alpha and beta isoforms in patients with NIDDM.

Activation of glycogen synthesis in skeletal muscle in response to insulin results from the combined inactivation of glycogen synthase kinase-3 (GSK-3) and activation of the protein phosphatase-1, changing the ratio between the inactive phosphorylated state of the glycogen synthase to the active dephosphorylated state. In a search for genetic defects responsible for the decreased insulin stimulated glycogen synthesis seen in patients with non-insulin-dependent diabetes mellitus (NIDDM) and their glucose-tolerant first-degree relatives we have performed mutational analysis of the coding region of the 2 isoforms of GSK-3alpha and GSK-3beta in 72 NIDDM patients and 12 control subjects. No structural changes were detected apart from a few silent mutations. Mapping of the GSK-3alpha to chromosome 19q13.1-13.2 and the GSK-3beta to chromosome 3q13.3-q21 outside known genetic loci linked to NIDDM further makes it unlikely that these genes are involved in the pathogenesis of common forms of NIDDM.

Alleles↗
PubMed1997

Genomic cloning and localization of CTAG, a gene encoding an autoimmunogenic cancer-testis antigen NY-ESO-1, to human chromosome Xq28.

CTAG was initially cloned from an esophageal squamous cell carcinoma cDNA expression library by immunoscreening with autologous patient's serum. CTAG mRNA is expressed in a proportion of human cancers in a lineage-nonspecific fashion, whereas its expression in normal tissues is restricted to testis and ovary only. This expression pattern suggests that the CTAG product (NY-ESO-1) is an aberrantly activated tumor antigen and can potentially be an antigenic target for tumor vaccination. In the present study, we isolated human genomic clones of CTAG and established its genomic organization. By somatic cell hybrid studies and fluorescence in-situ hybridization, we localized this gene to chromosome Xq28, a region that also contains members of MAGE, a gene family that encodes several immunogenic tumor antigens with the characteristic cancer/testis expression pattern.

Antigens, Neoplasm↗
PubMed1996

Allelic deletion mapping on chromosome 5 in human carcinomas.

We analysed allelic deletions on chromosome 5 in microdissected human non-small cell lung cancers. Thirty-four primary squamous cell carcinomas, 15 primary adenocarcinomas and five regional lymph node metastases were investigated for loss of heterozygosity (LOH) in chromosomal region 5p15-q21. The sites analysed included the APC tumor suppressor gene at 5q21, five polymorphic microsatellite markers and the putative tumor suppressor locus del-27, that was assigned to chromosomal region 5p13-12 by fluorescence in situ hybridization (FISH) analysis. Allelic deletions encompassed larger genomic regions more often in squamous cell carcinomas than in adenocarcinomas. The del-27 amd APC regions were identified as two distinct regions with the highest LOH frequencies within 5p15-q21. In squamous cell carcinomas LOH frequencies were 73% at the del-27 and 70% at the APC locus. In adenocarcinomas LOH at the del-27 and APC loci occurred in 38% of the informative cases. Allelic deletion of the APC gene and at the del-27 locus was also detected in the metastases. The results suggest involvement of at least two tumor suppressor genes on chromosome 5 in lung tumorigenesis.

Base Sequence↗
PubMed1996

Detection of the t(2;13) chromosomal translocation in alveolar rhabdomyosarcoma using the reverse transcriptase-polymerase chain reaction.

Rhabdomyosarcoma, a small-, round-cell tumor of skeletal muscle, is the most common soft tissue sarcoma found in children. A specific and unique chromosomal translocation, t(2;13)(q35;q14), has been described cytogenetically in a subset of these tumors and is most often associated with the alveolar histologic subtype. The cloning and sequencing of complementary DNA from fusion transcripts expressed by both cell lines and tumors have shown that this chromosomal translocation results in the fusion of the PAX3 gene on chromosome 2 with a member of the forkhead gene family, FKHR, on chromosome 13. To detect this genetic abnormality we have developed a sensitive method which relies on a reverse transcriptase-polymerase chain reaction with primers designed to be specific for the chromosome 2 and chromosome 13 sides of the translocation. The utility of this approach was tested by analyzing a series of rhabdomyosarcoma cell lines and tumor samples. The data demonstrate that the transcripts derived from the t(2;13) were restricted to tumors having features of the alveolar subtype and that they could be detected with greater ease and sensitivity than with cytogenetic analysis. This approach will facilitate a large-scale group effort to determine the frequency as well as the prognostic and diagnostic significance of this chromosomal rearrangement.

Cell Line↗
PubMed1996

Molecular cloning of the human leukotriene C4 synthase gene and assignment to chromosome 5q35.

BACKGROUND: Cysteinyl leukotrienes (LT) are mediators involved in inflammatory and allergic disorders LTC4 synthase catalyzes the first committed step in the synthesis of these inflammatory mediators, and its cellular distribution appears to be unique. MATERIALS AND METHODS: A human genomic library was screened by polymerase chain reaction (PCR) with primers that were designed based on the reported cDNA sequence for the LTC4 synthase gene. The gene was identified in one clone by Southern blotting of restriction enzyme digests, subcloning of fragments containing regions of interest, and DNA sequencing of these subclones. The transcription initiation site was determined by primer extension analysis. Chromosome location was determined by fluorescent in situ hybridization and screening of somatic cell hybrids by PCR. RESULTS: The LTC4 synthase gene is approximately 2.5 kb in length, consisting of five exons (136, 100, 71, 82, and 257 bp, respectively) and four introns (1,447, 102, 84, and 230 bp, respectively). Transcription initiation occurs at a single site 78 bp upstream of the coding region. The 5'-flanking region contains neither a TATA nor a CAAT box. The first 1 kb of the 5'-flanking region, however, contains putative DNA binding motifs for SP-1, AP-1, AP-2, ets factors, and CREB/ATF. A STAT binding motif is present in the first intron. The LTC4 synthase gene is located in the distal region of the long arm of chromosome 5 in 5q35. CONCLUSIONS: The LTC4 synthase gene does not contain elements of a typical regulated gene and may therefore contain novel regulatory elements. This gene is also located in a region on chromosome 5 that appears to play a role in allergic and inflammatory disorders, such as asthma.

Animals↗
PubMed1995

The IL-9 receptor gene (IL9R): genomic structure, chromosomal localization in the pseudoautosomal region of the long arm of the sex chromosomes, and identification of IL9R pseudogenes at 9qter, 10pter, 16pter, and 18pter.

Cosmids containing the human IL-9 receptor (R) gene (IL9R) have been isolated from a genomic library using the IL9R cDNA as a probe. We have shown that the human IL9R cDNA as a probe. We have shown that hte human IL9R gene is composed of 11 exons and 10 introns, stretching over approximately 17 kb, and is located within the pseudoautosomal region of the Xq and Yq chromosome, in the vicinity of the telomere. Analysis f the 5' flanking region revealed multiple transcription initiation sites as well as potential binding motifs for AP1, AP2, AP3, Sp1, and NF-kB, although this region lacks a TATA box. Using the human IL9R cosmid as a probe to perform fluorescence in situ hybridization, additional signals were identified in the subtelomeric regions of chromosomes 9q, 10p, 16p, and 18p. IL9R homologs located on chromosomes 16 and 10 were completely sequenced. Although they are similar to the IL9R gene (approximately 90% identity), none of these copies encodes a functional receptor: none of them contains sequences homologous to the 5' flanking region or exon 1 of the IL9R gene, and the remaining ORFs have been inactivated by various point mutations and deletions. Taken together, our results indicate that the IL9R gene is located at Xq28 and Yq12, in the long arm pseudoautosomal region, and that four IL9R pseudogenes are located on 9q34, 10p15, 16p13.3, and 18p11.3, probably dispersed as the result of translocations during evolution.

Alternative Splicing↗
PubMed1995

Chromosomal localization of mouse and human genes encoding the splicing factors ASF/SF2 (SFRS1) and SC-35 (SFRS2).

The mammalian SR-type splicing factors ASF/SF2 and SC-35 play crucial roles in pre-mRNA splicing and have been shown to shift splice site choice in vitro. We have mapped the ASF/SF2 gene in mice and humans and the SC-35 gene in mice. Somatic cell hybrid mapping of the human ASF/SF2 gene (SFRS1 locus) reveals that it resides on chromosome 17, and fluorescence in situ hybridization refines this localization to 17q21.3-q22. Recombinant inbred mapping of the mouse ASF/SF2 gene (Sfrs1 locus) and the mouse SC-35 gene (Sfrs2 locus) demonstrates that both genes are located in a part of mouse chromosome 11 that is homologous to human chromosome 17. Mapping of Sfrs1 using F1 hybrid backcross mice between the strains C57BL/6 and DDK places Sfrs1 very near the marker D11Mit38 and indicates that the ASF/SF2 gene is closely linked to the Ovum mutant locus.

Alternative Splicing↗
PubMed1995

A defined region of loss of heterozygosity at 11q23 in cutaneous malignant melanoma.

Karyotypic and molecular data indicate that genetic alterations of the long arm of chromosome 11 (11q) may be involved in malignant melanoma. To test this we analyzed 5 polymorphic microsatellite repeats on 11q using a PCR-based assay for loss of heterozygosity in normal and tumor tissues from 24 individuals with cutaneous malignant melanoma of various stages. Our findings indicate that a tumor suppressor gene that plays a role in malignant melanoma is located on the long arm of chromosome 11, likely within a 51 cM region at 11q23. Its loss appears to be a late event in tumor progression and may serve as an indicator for a less favorable clinical outcome.

Adult↗
PubMed1995

Localization of the human B-type natriuretic peptide precursor (NPPB) gene to chromosome 1p36.

Cardiac myocytes synthesize and secrete a family of peptide hormones with potent natriuretic, diuretic, and vasodilatory properties. These peptides are derived from precursor molecules that are encoded by two different genes, the atrial natriuretic peptide precursor A (NPPA) and the B-type natriuretic peptide or natriuretic peptide precursor B (NPPB). A human genomic clone for the NPPB gene was used to determine the chromosomal location of the NPPB gene. Analysis of Southern blot hybridization to DNAs from various somatic cell hybrids and fluorescence in situ hybridization allowed assignment of the NPPB locus to human chromosome 1p36. This location coincided with that of the NPPA locus; pulsed-field gel electrophoresis placed NPPA and NPPB within 50 kb of each other. This close chromosomal linkage, together with the conserved primary sequences and structural organization of the two natriuretic peptide precursor genes, suggests that the natriuretic peptide loci may have evolved from a common ancestor gene.

Animals↗
PubMed1995

Localization of short/branched chain acyl-CoA dehydrogenase (ACADSB) to human chromosome 10.

Short/branched chain acyl-CoA dehydrogenase, SBCAD (gene symbol ACADSB), is a member of the acyl-CoA dehydrogenase family of genes with activity toward the short/branched chain acyl-CoA derivatives as well as short/straight chain acyl-CoAs. Southern blot analysis of DNA from a panel of human/rodent somatic cell hybrids localized ACADSB to human chromosome 10, and fluorescence in situ hybridization experiments confirmed the chromosomal assignment and refined the subchromosomal localization to 10q25-q26.

Acyl-CoA Dehydrogenase↗
PubMed1995

Mutation and expression of TP53 in malignant melanomas.

Mutations of the TP53 gene are the most common genetic alterations in human malignancies. Overexpression of the p53 protein has been reported in high frequencies in all types of skin cancer. To determine the role of TP53 in the pathogenesis of malignant melanoma, we investigated the expression of p53 in 12 cell lines and 145 primary and metastatic lesions by immunohistochemistry. Overexpression of p53 was predominantly detected in the cytoplasm of the cells in 96 (66%) tumor and 12 (93%) cell lines. In contrast to findings in other tumor types, in melanomas immunoreactive cells were found in clusters or as scattered single cells. In primary melanomas, the frequency of p53 overexpression did not correlate with tumor thickness. Nucleotide sequencing of TP53 genes of 24 melanoma tumors/cell lines demonstrated point mutations in seven samples, all coding for mutant p53 protein species. The frequency of TP53 alterations of 20%-30% is lower than in other skin tumor types. Notably, immunohistochemistry was not a suitable method to distinguish overexpression of wild-type p53 from mutant species, since cell lines/tumors with TP53 mutations did not show distinctive staining patterns. The mutation pattern in six out of seven lesions was similar to that caused by ultraviolet light damage. This finding may be regarded a further indication for a pathogenetic role of UV light damage in at least a subgroup of malignant melanomas.

Antibodies, Monoclonal↗
PubMed1994

Loss of heterozygosity for 10q22-10qter in malignant melanoma progression.

Karyotypic and molecular data indicate that genetic events involving the chromosome region 10q22-10qter may be related to tumorigenesis in malignant melanoma. To test this we analyzed 10 polymorphic microsatellite repeats in the region 10q22-qter, using a polymerase chain reaction-based assay for loss of heterozygosity and DNA isolated from normal and tumor tissue from 26 individuals with malignant melanoma. The samples included 19 paired normal and malignant tissues representing various stages of melanoma as well as 7 cases in which samples from at least 2 different points in time during tumor progression were available. Our findings indicate that loss of heterozygosity of 10q22-10qter is a frequent event, that the observed loss of heterozygosity does not result from whole chromosome loss, and that it is associated with tumor progression. Finally, the appearance of new alleles in two of the tumors may indicate the involvement of DNA replication errors in melanoma analogous to such events in other tumor types.

Alleles↗