Translation initiation factor eIF-4G is immunogenic, overexpressed, and amplified in patients with squamous cell lung carcinoma.

BACKGROUND: Recently, the authors reported identification and cloning of several novel immunogenic antigens in squamous cell lung carcinoma. Of 14 corresponding genes, 9 mapped in an amplified chromosomal region with the gene for eIF-4G that was amplified most frequently. METHODS: Recombinant eIF-4G was expressed in E. coli and screened with sera from patients with squamous cell carcinoma of the lung and of the head and neck. Protein extracts from squamous cell carcinoma tissues were analyzed for eIF-4G expression by Western blot analysis. Mutation analysis was performed using an automatic DNA sequencer. RESULTS: The authors screened a spectrum of 33 heterologous sera from lung carcinoma patients and detected antibodies against eIF-4G in 5 of these sera (15%). They found no immune response to eIF-4G in 17 sera from squamous cell carcinoma tissues derived from the head and neck. In addition, no antibodies were found to eIF-4G in a group of 17 control sera from individuals without known tumor formation. Sequence analysis of the eIF-4G gave no indication of mutations. To analyze the expression of eIF-4G, a monoclonal antibody was used. Western blot analysis clearly showed overexpression in the tumor tissue compared with the corresponding normal lung tissues. CONCLUSIONS: The translation initiation factor eIF-4G is the first protein in which the gene shows amplification, has increased expression in a human tumor, and induces an immune response in patients. eIF-4G lends itself as a marker for the diagnosis of and possibly as a future therapeutic target in patients with squamous cell lung carcinoma.

Novel tankyrase-related gene detected with meningioma-specific sera.

In many meningiomas, alterations of chromosome 22 can be found, and the NF2 (neurofibromatosis type 2) gene, in particular, is of great interest as a putative gene involved in meningioma. Because the NF2 gene is not mutated in all meningiomas, additional genes may be involved. Instead of looking for alterations directly at the DNA level, we used the immune response of meningioma patients to identify immunogenic antigens that may be associated with the disease. We screened a fetal brain cDNA expression library with sera pools from different patients bearing meningioma classified according to the three WHO grades, using the serological identification of antigens by recombinant expression cloning immunological screening method. Here, we report the finding of a new tankyrase-related protein. We found 16 overlapping clones with homologies to tankyrase when we screened the library with the common-type meningioma sera pool and 2 such clones when we screened the library with the atypical meningioma sera. The anaplastic meningioma sera did not identify any tankyrase-related clones. We tested some of the newly identified clones with 13 single sera, 6 of which (37.5%) reacted positively with the tankyrase-related clones. In addition, we screened the tankyrase-related clone with six sera pools from individuals without obvious disease. Although 1 of 24 (4.2%) normal sera reacted with the tankyrase-related clone, we found a striking difference in the frequency of reactivity to this clone by sera from patients bearing tumors corresponding to the three WHO meningioma grades; common-type sera was the most frequently reactive. Northern blot analysis demonstrates expression of the novel tankyrase gene in two common-type meningiomas from patients with immune response.

Gene amplification at chromosome 1pter-p33 including the genes PAX7 and ENO1 in squamous cell lung carcinoma.

Gene amplification is a frequent event in lung cancer, specifically in squamous cell lung carcinoma. Recently, we reported amplifications on chromosomal bands 3q26.1-q26.3 with the genes BCHE and SLC2A2 amplified in 40% of squamous cell lung carcinomas. Here, we identified an amplified domain within chromosomal bands 1pter-p33 in squamous cell lung carcinoma using reverse chromosome painting. A panel of nine genes which have previously been assigned to region 1pter-p33 was tested for amplification using comparative PCR. The ENO1 gene that encodes enolase and the PAX7 gene that encodes a transcription factor were most frequently amplified. Specifically, the gene ENO1 was amplified in six and the gene PAX7 in five out of 37 cases which included both biopsies and paraffin-embedded tissues of squamous cell lung carcinomas. In total, we identified amplifications of at least one gene at bands 1pter-p33 in 10 out of 37 tumors (27%). Together, our data indicate that a novel and frequent amplification unit is present in squamous cell lung carcinoma with the center of the amplified domain in the vicinity of the genes PAX7 and ENO1.

Amplification of the genes BCHE and SLC2A2 in 40% of squamous cell carcinoma of the lung.

Gene amplification is a common genetic change in human cancer cells. Previously, we provided the first evidence for gene amplification at chromosome band 3q26 in squamous cell lung carcinoma. In this study, the following analyses were performed: (a) we evaluated biopsies and paraffin-embedded tissues of 16 additional squamous cell lung carcinomas for gene amplification using reverse chromosome painting. Of the 16 tumors, 3 tumors showed an amplification of the entire long arm of chromosome 3, and 3 tumors showed various amplifications on 3q, all of which involved chromosome band 3q26; (b) we tested eight genes encompassing region 3q25-qter in two different tumors to identify amplified genes on chromosome 3q. The genes SI, BCHE, and SLC2A2 were amplified in both tumors; and (c) we analyzed 15 additional paraffin-embedded tissues to determine the amplification frequency of these genes. Of the 15 squamous cell lung carcinomas, 6 showed amplification for at least 1 of the genes, with BCHE and SLC2A2 as the genes most frequently amplified. Together, our reverse chromosome painting data and our PCR analysis indicate gene amplification at 3q26 in 40% of all squamous cell lung carcinomas with BCHE and SLC2A2 as possible target genes of the amplification unit in squamous cell lung carcinoma.

DNA amplification on chromosome 3q26.1-q26.3 in squamous cell carcinoma of the lung detected by reverse chromosome painting.

Multiple genetic lesions have been reported in small cell lung carcinoma (SCLC), while considerably less information is available on squamous cell carcinoma (SCC). We used reverse chromosome painting to screen a total of nine SCCs for DNA amplifications. In three of the nine SCCs, hybridisation signals were found at chromosome region 3q26.1-q26.3, which does not contain any known oncogene. In one of the three SCCs, there were additional hybridisation signals at 1q, 5p and 6p21.1. The high frequency of a consistent amplification (3q26.1-q26.3) in SCC strongly indicates a novel gene at 3q26.1-q26.3 that is important in the pathology of SCC.

Chromosome specific c-DNA libraries: reduction of unspecific priming events by purification of heteronuclear RNA.

Chromosome specific c-DNA libraries greatly facilitate the isolation of disease associated genes which have been previously linked to particular chromosomes. Recently, several methods have been developed and employed for the isolation of transcribed sequences from specific human chromosomes and chromosome regions. Heteronuclear (hn) RNA from somatic human/rodent cell hybrids has been used as starting material to selectively prime the synthesis of human specific c-DNAs. A drawback of this method is the high number of rodent clones found in these chromosome specific c-DNA libraries. Here, we provide direct evidence that unspecific priming events account for the majority of these rodent clones. Using an Alu consensus primer hn-RNA human specific c-DNA libraries have been established and the specificity of Alu-priming has been evaluated. Using a variety of purification schemes for isolating hn-RNA we have significantly reduced the percentage of unspecific priming events. We also included a comparison of the hn-RNA yield from different somatic hybrids prior and after purification.

Human connexin43 gene locus, GJA1, sublocalized to band 6q21-->q23.2.

Gap junctions permit transfer of small molecules directly between cells. Several laboratories have mapped the human gap junction protein subunit, connexin43 (locus designation, GJA1), to overlapping regions of chromosome 6, localizing GJA1 to 6q14-->q24. We have sublocalized human GJA1 to a very small region. 6q21-->q23.2, using a human x rodent somatic cell hybrid mapping panel and a rat connexin43 cDNA probe.

Evidence for linear extrachromosomal elements mediating gene amplification in the multidrug-resistant J774.2 murine cell line.

Previous studies from our laboratory have demonstrated specific cytogenetic alterations accompanying development of colchicine resistance in the J774.2 murine cell line and in two sublines (J7.Cl-30 and J7.Cl-100). Although gene amplification is not observed in the parental J774.2 cell line, a approximately 35-fold amplification of the gene for p-glycoprotein (mdr) was noted in the J7.Cl-30 subline (770-fold CLCR) and a approximately 70-fold amplification in the J7.Cl-100 subline (2500-fold CLCR). In this study, we analyzed the localization and organization of the mdr gene. In the colchicine-resistant (CLCR) J7.Cl-30 subline, the p-glycoprotein domain was observed to reside on differently sized extrachromosomal elements. Our results indicate not only circular extrachromosomal elements but also linear extrachromosomal elements. By means of pulsed-field gel electrophoresis (PFGE), the sizes of the extrachromosomal elements were shown to be greater than 2,500 kilobase-pairs (kb), 800 kb, and 400 kb. In contrast, the J7.Cl-100 subline was characterized by the presence of homogeneously staining regions (HSRs). We have noted that with increasing colchicine resistance the extrachromosomal elements are replaced by HSRs. Our findings of linear elements that appear to be precursors of HSRs may offer a new way to interpret different theories of extrachromosomal gene amplification. The J7.Cl-30 cell line presents a unique system to analyze further the formation and structure of extrachromosomal elements.

Development and utilization of a somatic cell hybrid mapping panel to assign NotI linking probes to the long arm of human chromosome 6.

A somatic cell hybrid mapping panel that defines seven regions of the long arm and one region of the short arm of human chromosome 6 has been developed. Utilizing this panel, 17 NotI boundary clones from a NotI linking library were regionally assigned to the long arm of chromosome 6. The majority of these clones (11) were found to localize within band regions 6q24-q27. The nonuniform distribution of NotI sites may indicate a cluster of HTF islands and likely represents a coincidence of coding sequences in this region of chromosome 6. Cross-hybridization of these linking clones to DNA from other species (zoo blots) provides further evidence for transcribed sequences in 7 of the NotI clones. These NotI clones were also used to identify corresponding NotI fragments using pulsed-field gel electrophoresis, facilitating further physical mapping of this region. Finally, regional assignment of five polymorphic probes to the long arm of chromosome 6 is also presented. These hybrids and probes should facilitate the construction of a physical and genetic linkage map to assist in the identification of disease loci along chromosome 6.

Alu-PCR: characterization of a chromosome 6-specific hybrid mapping panel and cloning of chromosome-specific markers.

The Alu-polymerase chain reaction (Alu-PCR) was applied to selectively amplify DNA sequences from human chromosome 6 using a single primer (A1) directed to the human Alu consensus sequence. A specific amplification pattern was demonstrated for a panel of eight somatic cell hybrids containing different portions of chromosome 6. This PCR pattern permits the identification of submicroscopic DNA alterations and can be utilized as a reference for additional chromosome 6-specific hybrids. To obtain new chromosome 6-specific markers we established two libraries from PCR-amplified sequences using two somatic cell hybrids (MCH381.2D and 640-5A). Out of a total of 109 clones that were found to be chromosome 6 specific, 13 clones were regionally assigned. We also included a procedure that allows the isolation of chromosome 6-specific markers from hybrids that contain human chromosomes other than 6. Our results will contribute to the molecular characterization of chromosome 6 by fostering characterization of somatic cell hybrids and by the generation of new regionally assigned DNA markers.

nti glucocorticoid receptor transcripts lack sequences encoding the amino-terminal transcriptional modulatory domain.

Glucocorticoid induction of cell death (apoptosis) in mouse lymphoma S49 cells has long been studied as a molecular genetic model of steroid hormone action. To better understand the transcriptional control of glucocorticoid-induced S49 cell death, we isolated and characterized glucocorticoid receptor (GR) cDNA from two steroid-resistant nti S49 mutant cell lines (S49.55R and S49.143R) and the wild-type parental line (S49.A2). Our data reveal that nti GR transcripts encode intact steroid- and DNA-binding domains but lack 404 amino-terminal residues as a result of aberrant RNA splicing between exons 1 and 3. Results from transient cotransfection experiments into CV1 cells using nti receptor expression plasmids and a glucocorticoid-responsive reporter gene demonstrated that the truncated nti receptor exhibits a reduced transcriptional regulatory activity. Gene fusions containing portions of both the wild-type and the nti GR-coding sequences were constructed and used to functionally map the nti receptor mutation. We found that the loss of the modulatory domain alone is sufficient to cause the observed defect in nti transcriptional transactivation. These results support the proposal that glucocorticoid-induced S49 cell death requires GR sequences which have previously been shown to be required for transcriptional regulation, suggesting that steroid-regulated apoptosis is controlled at the level of gene expression.

Tumorigenicity in human melanoma cell lines controlled by introduction of human chromosome 6.

Chromosome banding analysis of human malignant melanoma has documented the nonrandom alteration of chromosome 6. To determine the relevance of chromosome 6 abnormalities in melanoma, a normal chromosome 6 was directly introduced into melanoma cell lines. The resulting (+6) microcell hybrids were significantly altered in their phenotypic properties in culture and lost their ability to form tumors in nude mice. The loss of the chromosome 6 from melanoma microcell hybrids resulted in the reversion to tumorigenicity of these cells in mice. The introduction of the selectable marker (psv2neo) alone into melanoma cell lines had no effect on tumorigenicity. These results support the idea that one or more genes on chromosome 6 may control the malignant expression of human melanoma.