Surfaceome profiling enables isolation of cancer-specific exosomal cargo in liquid biopsies from pancreatic cancer patients.

Background: Detection of circulating tumor DNA can be limited due to their relative scarcity in circulation, particularly while patients are actively undergoing therapy. Exosomes provide a vehicle through which cancer-specific material can be enriched from the compendium of circulating non-neoplastic tissue-derived nucleic acids. We carried out a comprehensive profiling of the pancreatic ductal adenocarcinoma (PDAC) exosomal 'surfaceome' in order to identify surface proteins that will render liquid biopsies amenable to cancer-derived exosome enrichment for downstream molecular profiling. Patients and methods: Surface exosomal proteins were profiled in 13 human PDAC and 2 non-neoplastic cell lines by liquid chromatography-mass spectrometry. A total of 173 prospectively collected blood samples from 103 PDAC patients underwent exosome isolation. Droplet digital PCR was used on 74 patients (136 total exosome samples) to determine baseline KRAS mutation call rates while patients were on therapy. PDAC-specific exosome capture was then carried out on additional 29 patients (37 samples) using an antibody cocktail directed against selected proteins, followed by droplet digital PCR analysis. Exosomal DNA in a PDAC patient resistant to therapy were profiled using a molecular barcoded, targeted sequencing panel to determine the utility of enriched nucleic acid material for comprehensive molecular analysis. Results: Proteomic analysis of the exosome 'surfaceome' revealed multiple PDAC-specific biomarker candidates: CLDN4, EPCAM, CD151, LGALS3BP, HIST2H2BE, and HIST2H2BF. KRAS mutations in total exosomes were detected in 44.1% of patients undergoing active therapy compared with 73.0% following exosome capture using the selected biomarkers. Enrichment of exosomal cargo was amenable to molecular profiling, elucidating a putative mechanism of resistance to PARP inhibitor therapy in a patient harboring a BRCA2 mutation. Conclusion: Exosomes provide unique opportunities in the context of liquid biopsies for enrichment of tumor-specific material in circulation. We present a comprehensive surfaceome characterization of PDAC exosomes which allows for capture and molecular profiling of tumor-derived DNA.

High prevalence of mutant KRAS in circulating exosome-derived DNA from early-stage pancreatic cancer patients.

Background: Exosomes arise from viable cancer cells and may reflect a different biology than circulating cell-free DNA (cfDNA) shed from dying tissues. We compare exosome-derived DNA (exoDNA) to cfDNA in liquid biopsies of patients with pancreatic ductal adenocarcinoma (PDAC). Patients and methods: Patient samples were obtained between 2003 and 2010, with clinically annotated follow up to 2015. Droplet digital PCR was performed on exoDNA and cfDNA for sensitive detection of KRAS mutants at codons 12/13. A cumulative series of 263 individuals were studied, including a discovery cohort of 142 individuals: 68 PDAC patients of all stages; 20 PDAC patients initially staged with localized disease, with blood drawn after resection for curative intent; and 54 age-matched healthy controls. A validation cohort of 121 individuals (39 cancer patients and 82 healthy controls) was studied to validate KRAS detection rates in early-stage PDAC patients. Primary outcome was circulating KRAS status as detected by droplet digital PCR. Secondary outcomes were disease-free and overall survival. Results: KRAS mutations in exoDNA, were identified in 7.4%, 66.7%, 80%, and 85% of age-matched controls, localized, locally advanced, and metastatic PDAC patients, respectively. Comparatively, mutant KRAS cfDNA was detected in 14.8%, 45.5%, 30.8%, and 57.9% of these individuals. Higher exoKRAS MAFs were associated with decreased disease-free survival in patients with localized disease. In the validation cohort, mutant KRAS exoDNA was detected in 43.6% of early-stage PDAC patients and 20% of healthy controls. Conclusions: Exosomes are a distinct source of tumor DNA that may be complementary to other liquid biopsy DNA sources. A higher percentage of patients with localized PDAC exhibited detectable KRAS mutations in exoDNA than previously reported for cfDNA. A substantial minority of healthy samples demonstrated mutant KRAS in circulation, dictating careful consideration and application of liquid biopsy findings, which may limit its utility as a broad cancer-screening method.

Mining the plasma proteome for disease applications across seven logs of protein abundance.

The current state of proteomics technologies has sufficiently advanced to allow in-depth quantitative analysis of the plasma proteome and development of a related knowledge base. Here we review approaches that have been applied to increase depth of analysis by mass spectrometry given the substantial complexity of plasma and the vast dynamic range of protein abundance. Fractionation strategies resulting in reduced complexity of individual fractions followed by mass spectrometry analysis of digests from individual fractions has allowed well in excess of 1000 proteins to be identified and quantified with high confidence that span more than seven logs of protein abundance. Such depth of analysis has contributed to elucidation of plasma proteome variation in health and of protein changes associated with disease states.

A silent, neutral substitution detected by reverse-phase high-performance liquid chromatography: hemoglobin Beirut.

A substitution of alanine for valine at position 126 in the beta-chain of hemoglobin was discovered in a hematologically normal adult male of Lebanese extraction. The variant beta-globin was initially observed and subsequently purified by reverse-phase high-performance liquid chromatography (HPLC). Reverse-phase HPLC was also used to isolate the variant tryptic peptide of beta-T13 that has alanine replacing valine at residue 126. The discovery of hemoglobin Beirut illustrates the usefulness of reverse-phase HPLC for the detection of neutral amino acid substitutions in proteins. The ability to detect neutral substitutions in undigested proteins is pertinent to the monitoring of genetic variation in human populations.

Impaired translational response and increased protein kinase PKR expression in T cells from lupus patients.

Activation of peripheral blood T cells results in a rapid and substantial rise in translation rates and proliferation, but proliferation in response to mitogen stimulation is impaired in systemic lupus erythematosus (SLE). We have investigated translation rates and initiation factor activities in T cells from SLE patients in response to activating signals. Activation by PMA plus ionomycin strongly increased protein synthesis in control T cells but not in T cells from SLE patients. The rate of protein synthesis is known to be strongly dependent on the activity of two eukaryotic translation initiation factors, eIF4E and eIF2alpha. We show that following stimulation, eIF4E expression and phosphorylation increased equivalently in control and SLE T cells. Expression of eIF4E interacting proteins - eIF4G, an inducer, and 4E-BP1 and 4E-BP2, two specific repressors of eIF4E function - and the phosphorylation level of 4E-BP1, were all identical in control and SLE T cells. In contrast, the protein kinase PKR, which is responsible for the phosphorylation and consequent inhibition of eIF2alpha activity, was specifically overexpressed in activated SLE T cells, correlating with an increase in eIF2alpha phosphorylation. Therefore, high expression of PKR and subsequent eIF2alpha phosphorylation is likely responsible, at least in part, for impaired translational and proliferative responses to mitogens in T cells from SLE patients.

Induction of the autoantigen proliferating cell nuclear antigen in T lymphocytes by a mycobacterial antigen.

Mycobacteria have been implicated in the pathogenesis of autoimmunity. To determine the potential effect of mycobacterial antigens on peripheral blood mononuclear cells (PBMC), we analyzed PBMC incubated with the acetone-precipitable fraction of Mycobacterium tuberculosis (APMT) for changes in cellular protein expression. Two-dimensional gel analysis showed induction of a 36-kD polypeptide identified as proliferating cell nuclear antigen (PCNA), a known autoantigen, after incubation with AP-MT. PCNA plays a role in cell proliferation and is expressed as a late growth regulated factor. However, its synthesis in response to AP-MT was induced as an early event. The early induction of PCNA was regulated at a posttranscriptional level and was restricted to T cells. Treatment of PBMC with known T cell mitogens, namely PHA, anti-CD3 antibodies, and staphylococcal superantigens failed to induce an early PCNA increase. The distinct characteristics of the AP-MT effect on PCNA expression suggest a separate mechanism of induction in response to AP-MT, compared with the late increase observed in response to mitogens. The induction of PCNA in response to mycobacterial antigens may represent a pathogenically relevant mechanism in autoimmunity.

Differential expression of Op18 phosphoprotein during human thymocyte maturation.

Op18 (also termed prosolin/stathmin) is a highly conserved 18-kD cytosolic phosphoprotein expressed in low levels in mature resting G0 lymphocytes, but induced in late G1 and S phases after entry into the cell cycle. In addition to its induction in normal proliferating lymphocytes, Op18 has been found to occur at high levels in acute leukemias and in neuroendocrine tissue. The presence and rapid phosphorylation of Op18 after stimulation of proliferating cells correlates with subsequent functional responses of the cells, and, therefore, Op18 has been suggested to play a key role in signal transduction. The pattern of expression of Op18 during lymphoid development is of interest in view of its high levels of expression in acute leukemias, representing cells arrested at an immature stage, thus raising the possibility that Op18 may be regulated differently in mature and immature lymphoid cells. We report here that immature human thymocytes bearing the cortical double positive phenotype (CD4+CD8+) constitutively express high levels of Op18 protein. In contrast, in mature single positive thymocytes (CD3+CD4+ or CD3+CD8+), Op18 protein is expressed at a lower level, comparable to that seen in peripheral blood T cells. Cell cycle analysis demonstrated that most of the cells in the double positive thymocyte population expressing high levels of Op18 were noncycling and arrested in G0. Furthermore, there was no correlation between Op18 levels and the proportion of cycling cells in double positive thymocyte populations isolated from different thymuses. Interestingly, although Op18 protein levels did not increase any further after mitogenic stimulation of double positive thymocytes, an increase in Op18 phosphorylation was observed, thus coupling of Op18 phosphorylation to cell activation remained intact. Our results show that during lymphoid maturation Op18 expression is uncoupled from cell proliferation. These data also suggest that the ordered expression of proliferation-associated genes seen in mature T cells may be disrupted during T cell maturation.

Proliferation-related expression of p19/nm23 nucleoside diphosphate kinase.

High level expression of the nm23-H1 gene, which encodes for a nucleoside diphosphate kinase, has been found to correlate with diminished metastasis in some tumors but not in others. We have previously identified the protein product of the nm23-H1 gene in two-dimensional electrophoretic gels and have designated it p19/nm23. In neuroblastoma, higher levels of p19/nm23, which are associated with amplification of the N-myc oncogene, large tumor mass, and metastasis, were observed in advanced stage tumors compared with limited stage disease. Because of the variable expression of nm23-H1 in different tumors, we have investigated the relationship between amounts of the protein and cell proliferation. The levels of p19/nm23 were compared between resting and mitotically stimulated normal human PBLs and in leukemia cells. The amount of p19/nm23 increased in normal lymphocytes in response to mitotic stimulation and paralleled the increase in DNA synthesis. In leukemia cells obtained from patients with different subtypes of acute leukemia, p19/nm23 levels were also increased relative to resting normal lymphocytes. Treatment of mitotically stimulated lymphocytes with cyclosporin, which inhibits proliferation, blocked the increase in p19/nm23; treatment of the leukemia cell line HL-60 with dimethylsulfoxide, which induces terminal differentiation, resulted in diminished levels of p19/nm23. Our data therefore provide evidence that nm23-H1 expression is related to cell proliferative activity.

Hsp27 expression in neuroblastoma: correlation with disease stage.

BACKGROUND: The 27-kd heat shock protein (Hsp27) is differentially expressed in some malignancies, including breast carcinoma, leukemia, and malignant fibrous histiocytoma. In breast carcinoma, a high-level expression of Hsp27 has been associated with shorter disease-free survival in patients with localized disease. PURPOSE: We have observed variable levels of Hsp27 among neuroblastoma tumors. Our aim in this study was to investigate the relationship between Hsp27 expression and stage of the disease and N-myc gene copy number. METHODS: We determined Hsp27 protein levels in 53 neuroblastoma tumors representing different stages of the disease and in 17 neuroblastoma cell lines by quantitative two-dimensional polyacrylamide gel electrophoresis (PAGE). We also performed statistical analysis of Hsp27 levels in relation to stage of the disease and to N-myc gene copy number. RESULTS: Increased Hsp27 expression in neuroblastomas was associated with limited stage disease and inversely correlated with N-myc gene amplification, a feature known to predict poor clinical outcome. An inverse correlation was also observed between N-myc gene amplification and Hsp27 protein levels among the neuroblastoma cell lines analyzed. Immunohistochemical staining of sections of neuroblastomas showed that Hsp27 was most prominently expressed in the cytoplasm of large ganglionic tumor cells present in neuronally differentiated areas of the tumors. Induction of neuronal differentiation in SMS-KCNR neuroblastoma cells using retinoic acid resulted in an increase in Hsp27. CONCLUSION: High level expression of Hsp27 in neuroblastoma is a feature of limited stage, differentiated tumors. IMPLICATION: Hsp27 may play a part in the biology of neuroblastomas with a favorable outcome.

Proteomics-based identification of protein gene product 9.5 as a tumor antigen that induces a humoral immune response in lung cancer.

We used a proteomic approach to identify proteins that commonly induce an antibody response in lung cancer. Sera from 64 newly diagnosed patients with lung cancer, 99 patients with other types of cancer, and 71 noncancer controls were analyzed for antibody-based reactivity against lung adenocarcinoma proteins resolved by two-dimensional PAGE. Unlike controls, autoantibodies against a protein identified by mass spectrometry as protein gene product 9.5 (PGP 9.5) were detected in sera from 9 of 64 patients with lung cancer. Circulating PGP 9.5 antigen was detected in sera from two additional patients with lung cancer, without detectable PGP 9.5 autoantibodies. PGP 9.5 is a neurospecific polypeptide previously proposed as a marker for non-small cell lung cancer, based on its expression in tumor tissue. Using A549 lung adenocarcinoma cell line, we have demonstrated that PGP 9.5 was present at the cell surface, as well as secreted. Thus, the findings of PGP 9.5 antigen and/or antibodies in serum of patients with lung cancer suggest that PGP 9.5 may have utility in lung cancer screening and diagnosis.

A minimal critical region of the 8p22-23 amplicon in esophageal adenocarcinomas defined using sequence tagged site-amplification mapping and quantitative polymerase chain reaction includes the GATA-4 gene.

The incidence of esophageal adenocarcinomas has increased greatly over the past 20 years. The genetic alterations associated with this disease, however, remain largely unknown. We identified recently a novel amplicon at 8p22-23 in esophageal adenocarcinomas using the restriction landmark genomic scanning two-dimensional gel technique. Four known genes within or near this amplicon were initially characterized. The cathepsin B (CTSB) gene was found to be amplified in 13% of esophageal tumors. CTSB was shown previously to be overexpressed without amplification in many other human cancers. An approach termed sequence tagged site-amplification mapping has been implemented in the present study, allowing the 8p22-23 amplicon to be narrowed from 12 cM to a <2-cM minimal amplified area located between markers D8S552 and D8S1759. The CTSB gene maps within this region. To identify other cancer-related candidate genes in this region, a positional candidate gene approach was subsequently applied to characterize this minimal critical region. An expressed sequence tag (EST), which was included in the minimal critical region, demonstrated both amplification and overexpression. This EST and the extended sequence from the EST were determined to be a novel sequence in the 3' untranslated region of the human GATA-4 gene. GATA-4, a member of a zinc finger transcription factor family, was confirmed to be amplified and overexpressed in esophageal adenocarcinomas and was localized within <0.5 kb from CTSB. Furthermore, amplification of 8p22-23 was detected in one of eight gastric cardia adenocarcinomas but was not observed in either human lung adenocarcinomas (n = 39) or in esophageal squamous cell carcinomas (n = 24). The relatively high frequency of the 8p22-23 amplification in esophageal (13.6%) and gastric cardia (12.5%) adenocarcinomas may indicate a specificity of this amplicon for tumors of gastroesophageal origin.

Identification and characterization of a 19q12 amplicon in esophageal adenocarcinomas reveals cyclin E as the best candidate gene for this amplicon.

Genomic DNA amplification in tumors is frequently associated with an increased gene copy number of oncogenes or other cancer-related genes. We have used a two-dimensional whole-genome scanning technique to identify gene amplification events in esophageal adenocarcinomas. A multicopy genomic fragment from a tumor two-dimensional gel was cloned, and genomic amplification encompassing this fragment was confirmed by Southern blot analysis. The corresponding DNA sequence was matched by BLAST to a BAC contig, which allowed the use of electronic-PCR to localize this amplicon to 19q12. Sequence tagged site-amplification mapping, an approach recently implemented in our laboratory (Lin, L. et al., Cancer Res., 60: 1341-1347,2000), was used to characterize the amplicon. Genomic DNA from 65 esophageal and 11 gastric cardia adenocarcinomas were investigated for 19q12 amplification using quantitative PCR at 11 sequence tagged site markers neighboring the cloned fragment. The amplicon was narrowed from >8 cM to a minimal critical region spanning <0.8 cM, between D19S919 and D19S882. This region includes the cyclin E gene. Fourteen expressed sequence tags (ESTs) covering the minimal region were then assayed for potential gene overexpression using quantitative reverse transcription-PCR. Seven of the selected ESTs were found to be both amplified and overexpressed. Among these seven ESTs, cyclin E showed the highest frequency of gene amplification and overexpression in the tumors examined, which allowed us to finalize the core-amplified region to <300 kb. These results indicate that cyclin E is the likely target gene selected by the amplification event at 19q12. The fact that cyclin E overexpression was found only in the amplified tumors examined indicates that gene amplification underlies the cyclin E gene overexpression. Our study represents the first extensive analysis of the 19q12 amplicon, and is the first to physically map the core-amplified domain to a region of <300 kb that includes cyclin E. Amplification of 19q12 was found neither in the 28 esophageal squamous cancers nor in the 39 lung adenocarcinomas examined but was observed in 13.8% of esophageal and 9.1% of gastric cardia adenocarcinomas.

Distinctive molecular profiles of high-grade and low-grade gliomas based on oligonucleotide microarray analysis.

Astrocytomas are heterogeneous intracranial glial neoplasms ranging from the highly aggressive malignant glioblastoma multiforme (GBM) to the indolent, low-grade pilocytic astrocytoma. We have investigated whether DNA microarrays can identify gene expression differences between high-grade and low-grade glial tumors. We compared the transcriptional profile of 45 astrocytic tumors including 21 GBMs and 19 pilocytic astrocytomas using oligonucleotide-based microarrays. Of the approximately 6800 genes that were analyzed, a set of 360 genes provided a molecular signature that distinguished between GBMs and pilocytic astrocytomas. Many transcripts that were increased in GBM were not previously associated with gliomas and were found to encode proteins with properties that suggest their involvement in cell proliferation or cell migration. Microarray-based data for a subset of genes was validated using real-time quantitative reverse transcription-PCR. Immunohistochemical analysis also localized the protein products of specific genes of interest to the neoplastic cells of high-grade astrocytomas. Our study has identified a large number of novel genes with distinct expression patterns in high-grade and low-grade gliomas.

The gene for the axonal cell adhesion molecule TAX-1 is amplified and aberrantly expressed in malignant gliomas.

The human TAX-1 gene encodes a Mr 135,000 glycoprotein that is transiently expressed on the surface of a subset of neurons during development and is involved in neurite outgrowth. The TAX-1 gene has been mapped to a region on chromosome 1 that has been implicated in microcephaly and the Van der Woude syndrome. Using restriction landmark genome scanning to search for amplified genes in gliomas, we found TAX-1 to be amplified in 2 high-grade gliomas among a group of 26 gliomas investigated. Real-time reverse transcription-quantitative PCR analysis detected high levels of TAX-1 mRNA in glial tumors, even in the absence of TAX-1 gene amplification. Immunohistochemical analysis revealed abundant levels of TAX-1 in neoplastic glial cells of glioblastoma multiforme tumors. Because glial tumors are highly invasive and in view of the role of TAX-1 in neurite outgrowth, we investigated the potential role of TAX-1 in glioma cell migration. Using an in vitro assay, we found that the migration of glioma tumor cells is profoundly reduced in the presence of either an anti-TAX-1 antibody or a TAX-1 antisense oligonucleotide. Our findings suggest that TAX-1 plays a role in glial tumorigenesis and may provide a potential target for therapeutic intervention.

Expression of the protein kinase PKR in modulated by IRF-1 and is reduced in 5q- associated leukemias.

The transcription factor IRF-1 (interferon regulatory factor 1) is an activator of type I interferon and interferon-inducible genes. IRF-1 manifests tumor suppressor activity. Its overexpression results in inhibition of cell growth, and deletions of the IRF-1 gene were demonstrated in a number of human leukemias and myelodysplasias. Although the mechanism by which IRF-1 affects cell growth is unknown, it is believed that IRF-1 activates a set of genes that negatively regulate cell growth. The double-stranded RNA-dependent protein kinase (PKR), which is an interferon-inducible gene, contains a promoter element for the binding of IRF-1 and exhibits antiproliferative properties. Consequently, we investigated the role of IRF-1 in PKR expression. Here, we show that in IRF-1-deficient embryonic fibroblasts, PKR expression is reduced relative to wild-type cells. This result predicts diminished expression of PKR as a potential consequence of deletion of the IRF-1 gene in human leukemias. We show that cells of the human leukemic U937 cell line contain a deletion of one IRF-1 gene and express low levels of PKR. We demonstrate that upregulation of IRF-1 expression in U937 cells by transfection is sufficient to induce PKR expression. We also found a marked reduction in the expression of PKR in blood samples from two patients with myelodysplasias, carrying a deletion of chromosome 5q, a locus to which IRF-1 was mapped. These results show that IRF-1 activates PKR expression and suggest that loss of one allele of the IRF-1 gene is sufficient to affect PKR expression. Therefore, PKR is a strong candidate for a mediator of the tumor suppressor activity of IRF-1.

A nucleoside diphosphate kinase A (nm23-H1) serine 120-->glycine substitution in advanced stage neuroblastoma affects enzyme stability and alters protein-protein interaction.

A high level of nucleoside diphosphate kinase A (NDPK A/nm23-H1) in neuroblastoma is associated with advanced stage disease. We have also found a serine 120-->glycine substitution in NDPK A and/or amplification of the nm23-H1 gene in advanced stage neuroblastomas. Serine 120, a highly conserved residue, is located in proximity to histidine 118 which forms a phosphorylated intermediate essential for NDPK activity. The effect of Ser120-->Gly substitution on the biochemical properties of NDPK A was investigated. Phosphate-transferase activity was lower in the recombinant mutant NDPK A and in the immunoprecipitated complex consisting of NDPK A and NDPK B prepared from a neuroblastoma tumor containing the mutation, relative to the wild-type. There was a significant decrease in the enzyme stability toward urea- or temperature-induced denaturation for the recombinant mutant NDPK A and in an immunoprecipitate from a tumor containing the mutation. Recombinant NDPK A containing the Ser120-->Gly mutation exhibited reduced hexameric and increased dimeric oligomerization relative to the wild-type. Moreover a 28 kDa cellular protein was detected, that co-precipitated with the mutant but not wild-type NDPK A. The altered properties of the mutant protein may have relevance to a role for NDPK A in neuroblastoma progression.

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 ubiquitous 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.

GAC1, a new member of the leucine-rich repeat superfamily on chromosome band 1q32.1, is amplified and overexpressed in malignant gliomas.

We have used two-dimensional electrophoresis of enzyme-digested genomic DNA to identify a novel gene GAC1, which maps at 1q32.1 and which is overexpressed in malignant gliomas in which it is amplified. GAC1 encodes a protein which belongs to the leucine-rich repeat superfamily. Amplification and overexpression of GAC1 was demonstrated in two of eight tumors where amplifications were previously evidenced by comparative genomic hybridization (one glioblastoma multiforme and one anaplastic astrocytoma), and in one of eight unselected glioblastomas multiforme. GAC1 exhibits sequence homology with other proteins which function as cell-adhesion molecules or as signal transduction receptor and is a likely candidate for the target gene in the 1q32.1 amplicon in malignant gliomas.

N-myc gene amplification in neuroblastoma is associated with altered phosphorylation of a proliferation related polypeptide (Op18).

We have recently identified and cloned the gene for a cytosolic polypeptide, designated oncoprotein 18 (Op18), which is expressed in acute lymphocytic leukemia and some solid tumors including neuroblastoma. Op18 is phosphorylated upon treatment of lymphoid cells with phorbol myristate acetate. We have proposed that unphosphorylated Op18 plays a role in cellular proliferation, and that its phosphorylated forms, namely Op18a and Op18b, are associated with diminished cell proliferation. In this study, we report that in neuroblastoma tumors, the phosphorylation of Op18 was substantially diminished with increasing N-myc gene copy number. Treatment of the neuroblastoma cell line SMS-KCNR, which contains 75 copies of the N-myc gene, with retinoic acid for ten days resulted in an increase in Op18 phosphorylation. Our findings provide evidence for distinct patterns of Op18 phosphorylation in neuroblastoma tumors with and without N-myc gene amplification.

Estimation of mutation rates based on the analysis of polypeptide constituents of cultured human lymphoblastoid cells.

A subclone of a human diploid lymphoblastoid cell line, TK-6, with consistently high cloning efficiency has been used to estimate the rates of somatic mutations on the basis of protein variation detected by two-dimensional polyacrylamide gel electrophoresis. A panel of 267 polypeptide spots per gel was screened, representing the products of approximately 263 unselected loci. The rate of human somatic mutation in vitro was estimated by measuring the proportion of protein variants among cell clones isolated at various times during continuous exponential growth of a TK-6 cell population. Three mutants of spontaneous origin were observed, giving an estimated spontaneous rate of 6 x 10(-8) electrophoretic mutations per allele per cell generation (i.e., 1.2 x 10(-7) per locus per cell generation). Following treatment of cells with N-ethyl-N-nitrosourea, a total of 74 confirmed variants at 54 loci were identified among 1143 clones analyzed (approximately 601,000 allele tests). The induced variants include 65 electromorphs which exhibit altered isoelectric charge and/or apparent molecular weight and nine nullimorphs for each of which a gene product was not detected at its usual location on the gel. The induced frequency for these 65 structural gene mutants is 1.1 x 10(-4) per allele. An excess of structural gene mutations at ten known polymorphic loci and repeat mutations at these and other loci suggest nonrandomness of mutation in human somatic cells. Nullimorphs occurring at three heterozygous loci in TK-6 cells may be caused by genetic processes other than structural gene mutation.