Barbara J. Lamb

Amplification and overexpression of the IGF2 regulator PLAG1 in hepatoblastoma

There is evidence that 8q amplification is associated with poor prognosis in hepatoblastoma. A previous comparative genomic hybridization analysis identified a critical region in chromosomal bands 8q11.2–q13. Using restriction landmark genomic scanning in combination with a virtual genome scan, we showed that this region is delineated by sequences within contig NT_008183 of chromosomal subbands 8q11.22–q11.23. A real‐time PCR–based genomic copy number assay of 20 hepatoblastomas revealed gain or amplification in this critical chromosomal region in eight tumors. The expression of four genes and expressed sequence tags (ESTs) within this newly defined region was assayed by real‐time reverse transcriptase polymerase chain reaction (RT‐PCR) in four tumors with and six tumors without gain or amplification. The PLAG1 oncogene was found to be highly expressed in all but one tumor compared to normal liver tissue. Furthermore, quantitative RT‐PCR revealed that the expression level of the developmentally regulated transcription factor PLAG1 was 3–12 times greater in hepatoblastoma tumors and cell lines compared to age‐matched normal liver and comparable to the expression in fetal liver tissue. PLAG1 has been shown be a transcriptional activator of IGF2 in other tumor types. Using luciferase reporter assays, we demonstrated that PLAG1 transactivates transcription from the embryonic IGF2 promoter P3, also in hepatoblastoma cell lines. Thus, our results provide evidence that PLAG1 overexpression may be responsible for the frequently observed up‐regulation of IGF2 in hepatoblastoma and therefore may be implicated in the molecular pathogenesis of this childhood neoplasia.

Co-amplification of a novel gene, NAG, with the N- myc gene in neuroblastoma

Substantial evidence implicates amplification of the N-myc gene with aggressive tumor growth and poor outcome in neuroblastoma. However some evidence suggests that this gene alone is not the sole determinant of outcome in N-myc amplified tumors. We have searched for genes that co-amplify with N-myc in neuroblastoma by means of two-dimensional analysis of genomic restriction digests. Using this approach, we have identified and cloned a novel genomic fragment which is co-amplified with N-myc in neuroblastomas. This fragment was mapped in close vicinity to N-myc on chromosome arm 2p24. It was amplified in 5/8 N-myc amplified neuroblastoma cell lines and in 9/13 N-myc amplified tumors. Using a PCR-based approach we isolated a 4.5 kb c-DNA sequence that is partly contained in the genomic fragment. The open reading frame of the cDNA encodes a predicted protein of 1353 amino acids (aa). The homology of the predicted protein, which we designated NAG (neuroblastoma amplified gene), to a C. elegans protein of as yet unknown function, and its ubiquious expression suggest that NAG may serve an essential function. By Northern blot analysis we showed that amplification of the cloned gene correlates with over-expression in neuroblastoma cell lines. Amplification and consequent over-expression of NAG may, therefore, contribute to the phenotype of a subset of neuroblastomas.

Amplification and overexpression of the L-MYC proto-oncogene in ovarian carcinomas

Gene amplification is an important mechanism of oncogene activation in various human cancers, including ovarian carcinomas (OvCas). We used restriction landmark genomic scanning (RLGS) to detect amplified DNA fragments in the genomes of 47 primary OvCas. Visual analysis of the RLGS gel images revealed several OvCa samples with spots of greater intensity than corresponding spots from normal tissues, indicating possible DNA amplification in specific tumors. Two primary tumors (E1 and S12) shared four high-intensity spots. A recently developed informatics tool termed Virtual Genome Scans was used to compare the RLGS patterns in these tumors with patterns predicted from the human genome sequence. Virtual Genome Scans determined that three of the four fragments localized to chromosome 1p34-35, a region containing the proto-oncogene L-MYC. Sixty-eight primary OvCas, including 40 analyzed by RLGS, were screened by quantitative polymerase chain reaction (PCR) for possible amplification of L-MYC. Ten tumors with increased L-MYC copy number were identified, including tumor E1, which showed an approximately 24-fold increase in copy number compared to normal DNA. Southern analysis of several tumors confirmed the quantitative PCR results. Using sequence tagged site (STS) markers flanking L-MYC, increased DNA copy number in tumor E1 was found to span the region flanking L-MYC between D1S432 and D1S463 ( approximately 3.1 Mb). Other tumors showed amplification only at the L-MYC locus. Using oligonucleotide microarrays, L-MYC was found to be more frequently overexpressed in OvCas than either c-MYC or N-MYC relative to ovarian surface epithelium. Quantitative reverse transcriptase-PCR analysis confirmed elevated L-MYC expression in a substantial fraction of OvCas, including nine of nine tumors with increased L-MYC copy number. The data implicate L-MYC gene amplification and/or overexpression in human OvCa pathogenesis.

Demethylation of repetitive DNA sequences in neuroblastoma

Altered genomic methylcytosine content has been described for a number of tumor types, including neuroblastoma. However, it remains to be determined for different tumor types whether specific loci or chromosomal regions are affected by a methylation change or whether the change is random. We have implemented a computer‐based approach for the analysis of two‐dimensional separations of human genomic restriction fragments. Through the use of methylation‐sensitive restriction enzymes, methylation differences in genomic DNA between tumor and normal tissues can be detected. We report the cloning and sequencing of two fragments detectable in two‐dimensional separations of genomic DNA of neuroblastomas. These fragments were found to be a part of repetitive units that exhibited demethylation in neuroblastoma relative to other tumor types. Our finding of a distinct pattern of methylation of repetitive units in neuroblastoma suggests that altered methylation at certain loci may contribute to the biology of this tumor. Genes Chromosom Cancer 17:234–244 (1996).

Distinct sequences on 11q13.5 and 11q23–24 are frequently coamplified with MLL in complexly organized 11q amplicons in AML/MDS patients

Amplification within chromosome arm 11q involving the mixed‐lineage leukemia gene (MLL) locus is a rare but recurrent aberration in acute myeloid leukemia and myelodysplastic syndrome (AML/MDS). We and others have observed that 11q amplifications in most AML/MDS cases have not been restricted to the chromosomal region surrounding the MLL gene. Therefore, we implemented a strategy to characterize comprehensively 11q amplicons in a series of 13 AML/MDS patients with MLL amplification. Analysis of 4 of the 13 cases by restriction landmark genomic scanning in combination with virtual genome scan and by matrix‐based comparative genomic hybridization demonstrated that the 11q amplicon in these four cases consisted of at least three discontinuous sequences derived from different regions of the long arm of chromosome 11. We defined a maximally 700‐kb sequence around the MLL gene that was amplified in all cases. Apart from the core MLL amplicon, we detected two additional 11q regions that were coamplified. Using fluorescence in situ hybridization (FISH) analysis, we demonstrated that sequences in 11q13.5 and 11q23–24 were amplified in 8 of 13 and 10 of 12 AML/MDS cases, respectively. Both regions harbor a number of potentially oncogenic genes. In all 13 cases, either one or both of these regions were coamplified with the MLL amplicon. Thus, we demonstrated that 11q amplicons in AML/MDS patients display a complex organization and have provided evidence for coamplification of two additional regions on the long arm of chromosome 11 that may harbor candidate target genes.

Familial molar tissues due to mutations in the inflammatory gene, NALP7, have normal postzygotic DNA methylation.

An imprinting disorder has been believed to underlie the etiology of familial biparental hydatidiform moles (HMs) based on the abnormal methylation or expression of imprinted genes in molar tissues. However, the extent of the epigenetic defect in these tissues and the developmental stage at which the disorder begins have been poorly defined. In this study, we assessed the extent of abnormal DNA methylation in two HMs caused by mutations in the recently identified 19q13.4 gene, NALP7. We demonstrate normal postzygotic DNA methylation patterns at major repetitive and long interspersed nuclear elements (LINEs), genes on the inactive X-chromosome, three-cancer related genes, and CpG rich regions surrounding the PEG3 differentially methylated region (DMR). Our data provide a comprehensive assessment of DNA methylation in familial molar tissues and indicate that abnormal DNA methylation in these tissues is restricted to imprinted DMRs. The known role of NALP7 in apoptosis and inflammation pinpoints previously unrecognized pathways that could directly or indirectly underlie the abnormal methylation of imprinted genes in molar tissues.

Combined restriction landmark genomic scanning and virtual genome scans identify a novel human homeobox gene, ALX3, that is hypermethylated in neuroblastoma

Restriction landmark genome scanning (RLGS) allows comparative analysis of several thousand DNA fragments in the genome and provides a means to identify CpG islands that are altered in tumor cells as a result of amplification, deletion, or methylation changes. We have developed a novel informatics tool, designated virtual genome scan (VGS), that makes it possible to predict automatically the sequence of fragments in RLGS patterns by matching to the human genome sequence. A combination of RLGS and VGS was utilized to identify changes of chromosome 1–derived fragments in neuroblastoma. A NotI‐EcoRV fragment was found to be absent frequently in neuroblastoma cell line RLGS patterns. Sequence prediction by VGS as well as cloning of the fragment showed that it contained a CpG island that is part of the human orthologue of the hamster homeobox gene Alx3. Expression analysis in a panel of human and mouse tissues showed predominant expression of ALX3 in brain tissue. Methylation‐sensitive sequence analysis of the promoter region in neuroblastoma cell lines indicated that methylation of specific sequences correlated with repression of the ALX3 gene. Expression was re‐induced after treatment with the methylation inhibitor 5‐aza‐2′‐deoxycytidine. Promoter methylation analysis of ALX3 in primary neuroblastoma tumors, using methylation‐sensitive polymerase chain reaction, found preferential ALX3 methylation in advanced‐stage tumors. The VGS approach we have implemented in combination with RLGS is useful for the identification of genomic CpG island–related methylation changes or deletions in cancer.

Cell cycle progression is associated with distinct patterns of phosphorylation of Op18

Op18 is a highly conserved major cytosolic phosphoprotein which has been implicated in signal transduction in a wide variety of cell types. Freshly isolated peripheral blood lymphocytes (PBL) constitutively express low levels of mostly unphosphorylated Op18. Following mitogenic stimulation of PBL, Op18 synthesis is induced at a time when cells are entering S-phase. In this study we have characterized Op18 phosphorylation during progression of freshly isolated PBL through the cell cycle. Transition from G0 to G1 following activation with OKT3 was associated with an increase in a phosphorylated form designated Op18c. Progression of cells through G1 into S resulted in an increase in phosphorylated Op18 forms, designated Op18a and Op18b, which paralleled new Op18 synthesis. Transition of cells into G2 + M resulted in the appearance of the more acidic phosphorylated forms Op18d and Op18e. Calphostin C, a specific inhibitor of protein kinase C, dramatically decreased all forms of phosphorylated Op18 in OKT3 treated Jurkat cells. Our results suggest that Op18 phosphorylation is mediated in part by PKC activation as well as by other kinases yielding different phosphorylated forms at specific stages of the cell cycle.

Immunoglobulin Production by Human Lymphocytoid Lines and Clones: Absence of Genic Exclusion

Immunoglobulin production was studied in established lines of normal human lymphocytes. Three lines which produced both immunoglobulin G and immunoglobulin M were cloned. Among the 25 immunoglobulin-producing clones, 23 produced both classes of immunoglobulins. These findings suggest that the phenomenon of genic exclusion does not hold for immunoglobulin production in lymphocytoid cells in culture.

Pleiotropic mutants of Chinese hamster cells with altered cytidine 5′-triphosphate synthetase

Following chemical mutagenesis and multiple-step indirect selection, four clones of Chinese hamster V79 cells were isolated which exhibited auxotrophy for thymidine, deoxycytidine, or deoxyuridine but not for cytidine or uridine. All were resistant to uridine, 3-deazauridine, 5-fluorouridine, thymidine, and cytosine arabinoside at concentrations that were toxic to wild-type V79 cells. The cytidine 5′-triphosphate (CTP) and deoxycytidine 5′-triphosphate (dCTP) pools in the mutants were expanded, but the uridine 5′-triphosphate (UTP) pool either decreased or remained unchanged relative to the wild-type level. Furthermore, since the parental cells appear to be deficient in dCMP deaminase activity and CTP (or one of its metabolites) has been shown to inhibit uridine 5′-diphosphate (UDP) reduction, an elevated CTP level should lead to the observed thymidine auxotrophy. It also explains the joint resistance of mutant clones to thymidine and cytosine arabinoside. The change in the ratio of intracellular dCTP to thymidine 5′-triphosphate (dTTP) may be responsible for the elevation in the rates of spontaneous mutations in these mutants.