Genomic Organization and Mutation Analysis ofp73in Oligodendrogliomas with Chromosome 1 p-Arm Deletions

p73, a protein having substantial structural and functional similarity to p53, has recently been identified and demonstrated to be a potential tumor suppressor. Its location on human chromosome 1p36.33 implicates p73 as a candidate for neuroblastoma. Like neuroblastoma, oligodendrogliomas also show a high frequency of deletions in chromosome 1p36.3. To determine whetherp73is a potential tumor suppressor gene involved in the development of oligodendrogliomas, we performed mutation analysis ofp73in oligodendrogliomas with chromosome 1 p-arm deletions. We first determined the genomic organization and the intron–exon boundary sequences of thep73gene by long PCR, vectorette PCR, and Southern hybridization. This gene spans about 65 kb with a large first intron. Primer pairs for the amplification of each of the 13 p73 encoding exons were designed in corresponding introns. The amplicons were then analyzed using the denaturing high-performance liquid chromatography system for mutations in thep73gene. Twenty oligodendroglioma samples with 1p36.3 deletions were screened, but no mutations were detected except for several polymorphisms. It is thus clear thatp73is not a candidate gene for oligodendroglioma despite its location in the frequently deleted 1p36.3 region.     

Mutation ofp53Is Not a Prerequisite for Immortalization of Human Fibroblasts by SV40 T Antigen

Thein vitrolife span of human cells is under genetic control and limited. Immortalized cells, however, can be obtained at a low frequency following expression of the SV40 T antigen gene though the steps that lead to immortality are not well understood. p53 has been implicated in cell cycle regulation and evidence suggests it may have a role in controlling life span in rodent and human cells. In this study, we investigated whether allelic loss or mutation ofp53was an essential step during SV40 immortalization leading to the appearance of immortal cell lines. The gross structure of thep53gene was examined in a primary fibroblast cell strain (1BR.3) and two SV40-immortalized derivatives, 1BRMT1 and 1BRgn2. There was no evidence for allelic loss of thep53gene during immortalization. The primary cells and the immortal derivatives all expressed authenticp53mRNAs, though the immortal cell lines had higher levels of expression. Sequence analysis of exons 5–8 did not detect mutations associated with the immortal phenotype. These data are consistent with SV40 immortalization being independent of genetic changes inp53.     

Characterization of newly established colorectal cancer cell lines: correlation between cytogenetic abnormalities and allelic deletions associated with multistep tumorigenesis

We have established a series of 20 colorectal cancer cell lines and performed cytogenetic and RFLP analyses to show that the recurrent genetic abnormalities of chromosomes 1, 5, 17 and 18 associated with multistep tumorigenesis in colorectal cancer, and frequently detected as recurrent abnormalities in primary tumours, are also retained in long-term established cell lines. Earlier studies by us and other investigators showed that allelic losses of chromosomes 1 and 17 in primary colorectal cancers predicted poorer survival for the patients (P = 0.03). We utilized the cell lines to identify specific chromosomal sites or gene(s) on chromosomes 1 and 17 which confer more aggressive phenotype. Cytogenetic deletions of chromosome 1p were detected in 14 out of the 20 (70%) cell lines, whereas allelic deletions for 1p using polymorphic markers were detected in 13 out of 18 (72%) informative cell lines for at least one polymorphic marker. We have performed Northern blotting, immunohistochemical staining (p53 mRNA, protein) and RFLP analysis using several probes including p53 and nm23. RFLP analysis using a total of seven polymorphic markers located on 17p and 17q arms showed allelic losses aroundthe p53 locus in 16 out of the 20 cell lines (80%), four of which were losses of thep53 locus itself. In addition, seven cell lines (out of nine informative cases) also showed losses of thenm23 gene, four with concurrent losses of thep53 locus, while the remaining three were homozygous. In addition, five out of seven cell lines withnm23 deletions were derived from hepatic metastatic tumours, and one cell line was obtained from recurrent tumour. A comparison between allelic deletions of 1p and functional loss ofnm23 gene revealed a close association between these two events in cell lines derived from hepatic metastasis. Following immunohistochemical staining, nine out of the twenty cell lines showed high levels (25–80%) of mutant p53, four showed intermediate levels (>20%), and seven had undetectable levels of the protein. Of these seven, four showed complete absence of mRNA. Of the remaining three cell lines one showed aberrant mRNA due to germline rearrangement of thep53 gene, whereas in two cell lines normal levels of mRNA were present. Nineteen of the 20 cell lines had normal germline configurations for thep53 gene, while one showed a rearrangement. These data suggest that functional loss ofp53 andnm23 genes accomplished by a variety of mechanisms may be associated with poor prognosis and survival. In addition, concurrent deletions of chromosome regions 17p, 17q and 1p were closely associated with high-stage hepatic metastatic disease. These cell lines with well-characterized genetic alterations and known clinical history provide an invaluable source of material for various biological and clinical studies relating to multistep colorectal tumorigenesis.     

Neuroblastoma epigenetics: From candidate gene approaches to genome-wide screenings

Neuroblastoma (NB) is a childhood tumor originating from sympathetic nervous system cells. Although recently new insights into genes involved in NB have emerged, the molecular basis of neuroblastoma development and progression still remains poorly understood. The best-characterized genetic alterations include amplification of the proto-oncogene MYCN, ALK activating mutations or amplification, gain of chromosome arm 17q and losses of 1p, 3p, and 11q. Epigenetic alterations have been described as well: caspase-8 (CASP8) and RAS-association domain family 1 isoform A (RASSF1A) DNA-methylation are important events for the development and progression of neuroblastoma. In total, there are about 75 genes described as epigenetically affected in NB cell lines and/or NB primary samples. These epigenetic alterations were either found using a candidate gene approach or based on the analysis of genome-wide screening techniques. This review gives an extensive overview of all epigenetic changes described in NB as of today, with a main focus on both prognostic use and the potential of genome-wide techniques to find epigenetic prognostic biomarkers in NB. We summarize the key findings so far and the state-of-the-art of the upcoming methods at a unique time frame in the transition towards combined genome wide chromatin immune-precipitation (ChIP) and DNA sequencing techniques.     

The Gene Encoding the Catalytic Subunit Cα of cAMP-Dependent Protein Kinase (Locus PRKACA) Localizes to Human Chromosome Region 19p13.1

We have determined the chromosomal localization of the gene for the catalytic subunit Cα of cAMP-dependent protein kinase (locus PRKACA) to human chromosome 19 using polymerase chain reaction (PCR) and Southern blot analysis of two different somatic cell hybrid mapping panels. In addition, PCR analysis of a chromosome 19 mapping panel revealed the presence of a human Cα-specific amplification product only in cell lines containing the region 19p13.1 to 19q12. Finally, two-color fluorescencein situhybridization to metaphase chromosomes using the human Cα cDNA and human chromosome 19 inter-Alu-PCR product as probes localized the human Cα gene to chromosome region 19p13.1.     

Isolation of Two Novel WNT Genes, WNT14 and WNT15, One of Which (WNT15) Is Closely Linked to WNT3 on Human Chromosome 17q21

TheWntgene family consists of at least 15 structurally related genes that encode secreted extracellular signaling factors. Wnt proteins function in a range of critical developmental processes in both vertebrates and invertebrates and are implicated in regulation of cell growth and differentiation in certain adult mammalian tissues, including the mammary gland. We have isolated a number of WNT sequences from human genomic DNA, two of which, designated WNT14 and WNT15, represent novel members of theWntgene family. We also isolated WNT sequences from human mammary cDNA and present evidence that WNT13 is expressed in human breast tissue, in addition to those previously described. WNT14 and WNT15 appear to have originated from an ancestral branch of theWntgene family that also includes theWnt9sequences found in jawless and cartilaginous fishes. AWnt14cDNA was also isolated from chicken and a partialWnt15sequence from mouse. We show that human WNT14 maps to chromosome 1 and that WNT15 maps distal to BRCA1 on chromosome 17q21, where it lies within 125 kb of another WNT family member, WNT3.     

Epigenetic inactivation and aberrant transcription of <Emphasis Type="Italic">CSMD1</Emphasis> in squamous cell carcinoma cell lines

Background  

The p23.2 region of human chromosome 8 is frequently deleted in several types of epithelial cancer and those deletions appear to be associated with poor prognosis. Cub and Sushi Multiple Domains 1 (CSMD1) was positionally cloned as a candidate for the 8p23 suppressor but point mutations in this gene are rare relative to the frequency of allelic loss. In an effort to identify alternative mechanisms of inactivation, we have characterized CSMD1 expression and epigenetic modifications in head and neck squamous cell carcinoma cell lines.     

Molecular analysis of the MHC class II region inDR4, DR7, andDR9 haplotypes

Within the class 11 region of the major histocompatibility complex (MHC) the amount of DNA in theDR-DQ interval has been shown to be haplotype dependent, with those carrying the DR4, DR7, and DR9 specificities having been reported to contain 110–160 kilobases (kb) more DNA than haplotypes carrying the DR3 specificity. Certain subtypes of haplotypes carrying particular DR specificities are more closely associated with autoimmune diseases than others. With the prospect of the DNA perhaps containing a disease susceptibility locus, we have mapped eight DR4 and two DR7 homozygous cell lines and a DR7/9 heterozygous cell line together with a control DR3 cell line using pulsed field gel electrophoresis (PFGE) with the enzymesBss H II,Pvu I, andNot I/Nru I. Our results, however, show that the presence and amount of the extra DNA is constant irrespective of the subtype. We have also tried to narrow down the position of insertion of the extra DNA using eight further rare-cutting enzymes but, due to the polymorphic nature of sites and/or differences in methylation in this region, it was not possible to refine it further than between DRA and DQA1/B1. This polymorphic nature of theDR-DQ region is unusual, considering the uniformity of rare cutter sites that has been observed within the rest of the class II, and class III, regions. The presence of this, and other, haplotype dependent variations in the DNA content of theDR subregion may be important with respect to recombination and will be particularly interesting if the additional DNA is found to contain novel genes.     

Molecular characterization of the genomic region harboring the BYDV-resistance geneBdv2 in wheat

Barley yellow dwarf virus (BYDV) can cause significant losses of wheat worldwide. The long arm segment ofThinopyrum intermedium chromosome 7Ai#1 carrying the BYDV resistance geneBdv2 was translocated to the distal region of the long arm of wheat chromosome 7D in translocation line Yw642. In this study, 40 wheat EST sequences located in the distal region of 7DL were explored to identify specific PCR markers for theBdv2 region on the basis of the homoeologous relationship between wheat chromosome 7D and Th.intermedium chromosome 7Ai# 1. Our results revealed 8 novel EST-PCR markers specific to theBdv2 region, including 5 EST-STS markers of BE404744, BE498985, BE591497, BG606695 and BQ161842, and 3 EST-SSCP markers of BE404953, BG312663 and BE498985. These EST-PCR markers could distinguishBdv2 from another BYDV-resistance gene located onTh.intermedium chromosome 2Ai-2. These specific bands for theBdv2 region were further cloned and sequenced. The sequencing analysis indicated that the specific sequences for theBdv2 region were highly homologous with the original wheat EST sequences that were used to design primers, and encode respectively a protein kinase, P450, centrin, transducin, and a hypothetical protein. This study created a starting point for eventual cloning of theBdv2 gene and understanding the defense mechanism.     

High-Resolution Genetic Map of the Human Glutamyl Aminopeptidase Gene (ENPEP)

The murine B-lymphocyte differentiation antigen BP-1/6C3 has been identified as glutamyl aminopeptidase (EAP), the gene symbol for which isENPEP.Using genomic DNA encoding for human EAP as a probe, we identified theENPEPgene location on human chromosome 4q25 by polymerase chain reaction analysis of a human/rodent somatic cell hybrid mapping panel and by fluorescencein situhybridization. Using a radiation hybrid panel, the gene order aroundENPEPwas determined to be centromere–D4S1236–(570 kb)–ENPEP–(210 kb)–D4S262–(270 kb)–D4S953–(270 kb)–D4S474–(570 kb)–IF. The linkage ofENPEPto complement factor I (IF) confirms the human chromosome band 4q25 localization predicted from the chromosomal location of murineENPEP.HumanENPEPthus provides an additional marker for the long arm of chromosome 4 that should facilitate studies of this genomic region.     

Human Rod Monochromacy: Linkage Analysis and Mapping of a Cone Photoreceptor Expressed Candidate Gene on Chromosome 2q11

We have performed linkage analysis in eight families with rod monochromacy, an autosomal recessively inherited condition with complete color blindness. Significant linkage was found with markers located at the pericentromeric region of chromosome 2. A maximum lod score of 5.36 was obtained for marker D2S2333 at θ = 0.00. Mapping of meiotic breakpoints localized the disease gene between markers D2S2187 and D2S2229. Homozygosity for a number of subsequent markers indicating identity by descent was found in two families and provides evidence for a further refinement of the locus proximal to D2S373. This defines an interval of ≈3 cM covering theACHM2locus for rod monochromacy. Radiation hybrid mapping of theCNGA3gene encoding the α-subunit of the cGMP gated cation channel in human cone photoreceptors resulted in a maximum lod score of 16.1 with marker D2S2311 combined with a calculated physical distance of 6.19cR_10,000. Screening of the CEPH YAC library and subsequent STS mapping indicated the physical order cen–D2S2222–D2S2175–(D2S2187/D2S2311)–qtel ofmarkers on 2q11 and showed that theCNGA3gene maps most closely to D2S2187 and D2S2311. These data indicate that theCNGA3gene maps within the critical interval of theACHM2locus for rod monochromacy and thus is a candidate gene for this disease.     

The Gene Encoding Protein Kinase SEK1 Maps to Mouse Chromosome 11 and Human Chromosome 17

We report the mapping of the human and mouse genes encoding SEK1 (SAPK/ERK kinase-1), a newly identified protein kinase that is a potent physiological activator of the stress-activated protein kinases. The human SERK1 gene was assigned to human chromosome 17 using genomic DNAs from human–rodent somatic cell hybrid lines. A specific human PCR product was observed solely in the somatic cell line containing human chromosome 17. The mouseSerk1gene was mapped to chromosome 11, closely linked toD11Mit4,using genomic DNAs from a (C57BL/6J ×Mus spretus)F₁×M. spretusbackcross.     

Retrovirus-mediated gene transfer of the cytokine genes interleukin-1beta and tumor necrosis factor-alpha into human neuroblastoma cells: consequences for cell line behavior and immunomodulatory properties

We have investigated the value of a gene therapy approach for neuroblastoma (NB), based on retroviral transduction of the IL-1beta or TNF-alpha cytokine genes into human NB lines. Secretion of the corresponding cytokine, was demonstrated in all lines, although with considerable quantitative variations. Cytokine gene expression significantly reduced the proliferation index (p = 0.0001); this effect was associated with either terminal neuronal (one TNF-alpha line) or fibroblast-like differentiation (two IL-1beta lines), leading to growth arrest after a few weeks. Cell surface levels of CD54 and HLA class II remained unaffected, but HLA class I (p < 0.001) and CD58 expression (p = 0.01) increased on SKNSH after TNF-alpha gene transfer. Mononuclear cells from normal allogeneic donors cocultured with both IL-1beta (p < 0.001) and TNF-alpha lines (p < 0.01), showed a significant increase in the proportion of activated T cells (CD3+DR+); however, their cytotoxicity and proliferation rate remained unchanged. Immunotherapy of neuroblastoma will require identification of transduced lines in which cytokine secretion induces phenotypic changes in such a way as to augment their likely immunomodulatory properties without impeding cell growth. Because of the limited efficacy of IL-1beta or TNF-alpha gene transfer alone, further studies should focus on combination with other immunomodulatory agents, to improve their potential efficacy in neuroblastoma.