C-myc Gene Locus Research Articles

Clinical Impact of C-myc Oncogenic Diversity on Solid and Lymphoid Malignancies.

Onset and progression of malignant tumors is a multistep process including a variety of gross chromosomal and specific genes' deregulation. Among oncogenes that are frequently altered in solid and also in hematological malignancies, the C-myc (gene locus: 8q24.21) plays a pivotal role. C-myc is a proto-oncogene encoding for a nuclear phosphoprotein implicated in cell cycle progression, apoptosis and cellular differentiation and transformation. The purpose of the current molecular review was to explore the differences of C-myc oncogenic activity in solid and lymphoid malignancies that modify its clinical impact on them. A systematic review of the literature in the international database PubMed was carried out. The year 2010 was set as a prominent time limit for the publication date of articles in the majority of them, whereas specific references of great importance and historical value in the field of C-myc gene discovery and analysis were also included. The following keywords were used: C-myc, oncogene, signaling pathway, malignancies, carcinoma, lymphoma. A pool of 43 important articles were selected for the present study at the basis of combining molecular knowledge with new targeted therapeutic strategies. C-myc oncogene demonstrates two different mechanisms of deregulation: amplification, mutation and translocation patterns. These particular aspects of gene alteration are unique for solid and non-solid (hematological) malignancies, respectively. C-myc is characterized by diversity regarding its deregulation mechanisms in malignancies derived from different tissues. C-myc translocation is sporadically combined with amplification ("complicon" formation) or mutations creating exotic genetic signatures. This "bi-phasic" C-myc deregulation model in the corresponding malignant tumor categories clinically affects the corresponding patients, also modifying the targeted therapeutic strategies on them.

Association between C-myc and K-ras gene polymorphisms and non-Hodgkin lymphoma.

To explore the association between c-myc and K-ras gene polymorphisms and non-Hodgkin lymphoma (NHL). A total of 200 NHL patients in our hospital in the past 3 years were collected as disease group, while 200 healthy people were taken as control group. The genomic deoxyribonucleic acid (DNA) in the peripheral blood was extracted in both groups, amplified via Polymerase Chain Reaction (PCR) and sent to the company for the detection of c-myc and K-ras gene polymorphisms. The expressions of c-myc and K-ras were detected via Reverse Transcription-quantitative PCR (RT-qPCR), and the levels of clinical indexes hemoglobin (Hb), platelet (PLT) and lactate dehydrogenase (LDH) were determined in the Laboratory Department. The allele distribution at c-myc gene locus rs121918684 was different between control group and disease group (p=0.000), and the G allele frequency was 202 (0.505) in the control group and 263 (0.657) in the disease group. In the disease group, the GG genotype frequency at c-myc gene locus rs121918684 [97 (0.485)], the CC genotype frequency at rs775522201 [98 (0.490)], and the GA genotype frequency at K-ras gene locus rs1137188 [127 (0.635)] were all significantly higher than those in the control group (p=0.000, p=0.002, p=0.011). In the disease group, the frequency of recessive model GC+CC (p=0.003), heterozygous model GC (p=0.035), and homozygous model CC (p=0.037) at c-myc gene locus rs121918684 was significantly lower than that in the control group, and the frequency of recessive model CT+TT (p=0.046) at c-myc gene locus rs775522201 was also markedly lower than that in the control group. The haplotype frequency of c-myc CC (p=0.000), GC (p=0.000), and GT (p=0.018) in the disease group was different from that in the control group. Moreover, the CT genotype at c-myc gene locus rs775522201 was remarkably correlated with the c-myc gene expression, and the gene expression was markedly increased in the disease group. The TT genotype at K-ras gene locus rs12245 was correlated with the K-ras gene expression, and the gene expression was notably increased in the disease group. There was an association between GG genotype at c-myc gene locus rs121918684 and LDH level (p=0.000), between CT genotype at c-myc gene locus rs775522201 and PLT level (p=0.002), and between AA genotype at K-ras gene locus rs1137188 and Hb level (p=0.003). The c-myc and K-ras gene polymorphisms are associated with susceptibility to NHL, gene expression and levels of Hb, PLT, and LDH.

TP53 Gene Mutations in Tumor Cells of Patients with Aggressive B-Cell Lymphomas

Background. TP53 gene mutations impede cell apoptosis and lead to additional oncogenic events contributing to tumor progression. Aim. To assess TP53 gene mutation rate in patients with high-grade B-cell lymphoma double-hit (HGBCL DH) and not otherwise specified (HGBL NOS); to analyse its relationship with disease prognosis. Materials & Methods. Retrospective materials from medical data of 10 HGBL DH and 26 HGBL NOS patients were analyzed. Median follow-up was 26.5 months (range 0.6-160.9 months). Selection was based on the presence of available biological materials (paraffin blocks) for Sanger sequencing of TP53 gene from exon 5 to exon 8 (encoding DNA-binding domain of TP53 gene). FISH analysis of the tumor was performed in all patients to identify translocations involving c-MYC/8q24, BCL2/18q21, and BCL6/3q27 gene locus. To analyze differences between groups χ<sup>2</sup> and Mann-Whitney tests were applied. Univariate event analysis (Kaplan-Meier and log-rank tests) and Cox regression analysis were used to assess the influence of molecular markers on the disease prognosis. Results. TP53 gene mutations in lymphoma cells were found in 13 (36 %) out of 36 patients, 10 (77 %) out of 13 mutations were pathogenic. In 8 out of 10 patients with TP53 mutations c-MYC/8q24 gene translocation was identified. Groups with wild (TP53-WT) and mutant (TP53-MUT) types of TP53 gene were similar in terms of main clinical characteristics. Patients with TP53-MUT in tumor cells showed worse 3-year overall survival (OS) compared with the group without TP53-MUT (30 % vs. 73 %; p = 0.026) as well as higher probability of disease progression in the period of 3 years (66 % vs. 15 %; p = 0.004). In multivariate analysis significant OS predictor was the presence of TP53 mutation (p = 0.006). Relapse/ progression probability was higher in combined cases of TP53 mutation and translocation involving c-MYC gene locus (p = 0.0003). Conclusion. Translocation involving c-MYC gene along with TP53 gene mutation in tumor cells can serve as a criterion for dividing HGBL DH and HGBL NOS patients into different lymphoma relapse/progression risk groups.

Abstract 4135: c-MYC drives a subset of high-risk pediatric neuroblastoma and is activated through mechanisms including enhancer hijacking and focal enhancer amplification

Abstract The amplified MYCN gene serves as an oncogenic driver in approximately 20% of high-risk pediatric neuroblastomas. Here we show that c-MYC itself is a potent transforming gene in a separate subset of high-risk neuroblastoma cases (~10%), based on (i) its upregulation by focal enhancer amplification or genomic rearrangements leading to enhancer hijacking, and (ii) its ability to transform neuroblastoma precursor cells in a transgenic animal model. The aberrant regulatory elements associated with oncogenic c-MYC activation include focally amplified distal enhancers or translocation of highly active enhancers from other genes to within topologically associating domains containing the c-MYC gene locus. The clinical outcome for patients with high levels of c-MYC expression is virtually identical to that of patients with amplification of the MYCN gene, a known high-risk feature of this disease. Together, these findings establish c-MYC as a bona fide oncogene in a clinically significant group of high risk childhood neuroblastomas. Citation Format: Mark W. Zimmerman, Yu Liu, Shuning He, Adam D. Durbin, Brian J. Abraham, John Easton, Ying Shao, Xu Beisi, Shizhen Zhu, Xiaoling Zhang, Zhaodong Li, Nina Weichert-Leahey, Richard A. Young, Jinghui Zhang, A. Thomas Look. c-MYC drives a subset of high-risk pediatric neuroblastoma and is activated through mechanisms including enhancer hijacking and focal enhancer amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4135.

Signal transducer and activator of transcription STAT5 is recruited to c-Myc super-enhancer.

Backgroundc-Myc has been proposed as a putative target gene of signal transducer and activator of transcription 5 (STAT5). No functional STAT5 binding site has been identified so far within the c-Myc gene locus, therefore a direct transcriptional regulation by STAT5 remains uncertain. c-Myc super-enhancer, located 1.7 Mb downstream of the c-Myc gene locus, was recently reported as essential for the regulation of c-Myc gene expression by hematopoietic transcription factors and bromodomain and extra-terminal (BET) proteins and for leukemia maintenance. c-Myc super-enhancer is composed of five regulatory regions (E1–E5) which recruit transcription and chromatin-associated factors, mediating chromatin looping and interaction with the c-Myc promoter.ResultsWe now show that STAT5 strongly binds to c-Myc super-enhancer regions E3 and E4, both in normal and transformed Ba/F3 cells. We also found that the BET protein bromodomain-containing protein 2 (BRD2), a co-factor of STAT5, co-localizes with STAT5 at E3/E4 in Ba/F3 cells transformed by the constitutively active STAT5-1*6 mutant, but not in non-transformed Ba/F3 cells. BRD2 binding at E3/E4 coincides with c-Myc transcriptional activation and is lost upon treatment with deacetylase and BET inhibitors, both of which inhibit STAT5 transcriptional activity and c-Myc gene expression.ConclusionsOur data suggest that constitutive STAT5 binding to c-Myc super-enhancer might contribute to BRD2 maintenance and thus allow sustained expression of c-Myc in Ba/F3 cells transformed by STAT5-1*6.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-016-0063-y) contains supplementary material, which is available to authorized users.

CMYC expression in infiltrating gliomas: associations with IDH1 mutations, clinicopathologic features and outcome

Gliomas are among the most frequent adult primary brain tumors. Mutations in IDH1, a metabolic enzyme, strongly correlate with secondary glioblastomas and increased survival. cMYC is an oncogene also implicated in aberrant metabolism, but its prognostic impact remains unclear. Recent genotyping studies also showed SNP variants near the cMYC gene locus, associate with an increased risk for development of IDH1/2 mutant gliomas suggesting a possible interaction between cMYC and IDH1. We evaluated nuclear cMYC protein levels and IDH1 (R132H) by immunohistochemistry in patients with oligodendroglioma/oligoastrocytomas (n = 20), astrocytomas (grade II) (n = 19), anaplastic astrocytomas (n = 21) or glioblastomas (n = 111). Of 158 tumors with sufficient tissue, 110 (70 %) showed nuclear cMYC immunopositivity--most frequent (95 %, χ(2) p = 0.0248) and intense (mean 1.33, ANOVA p = 0.0179) in anaplastic astrocytomas versus glioblastomas (63 %) or low grade gliomas (74 %). cMYC expression associated with younger age as well as p53 immunopositivity (OR = 3.6, p = 0.0332) and mutant IDH1 (R132H) (OR = 7.4, p = 0.06) among malignant gliomas in our cohort. Independent analysis of the publically available TCGA glioblastoma dataset confirmed our strong association between cMYC and mutant IDH1 expression. Both IDH1 (R132H) and cMYC protein expression were associated with improved overall survival by univariate analysis. However, cMYC co-expression associated with shortened time to malignant transformation and overall survival among IDH1 (R132H) mutants in both univariate and multivariate analyses. In summary, our findings suggest that cMYC may be associated with a unique clinicopathologic and biologic group of infiltrating gliomas and help mediate the malignant transformation of IDH1 mutant gliomas.

Tumor-specific histone signature and DNA methylation in multiple myeloma and leukemia cells

Understanding the epigenetics of tumor cells is of clinical significance for the treatment of cancer, and thus, chemists have focused their efforts on the synthesis of new generation of inhibitors of histone deacetylases (HDACs) or methylation-specific enzymes as novel important anti-cancer drugs. Here, we tested whether the histone signature and DNA methylation in multiple myeloma (MM) and leukemia cells is tumor-specific as compared with that in non-malignant lymphoblastoid cells. We observed a distinct histone signature in c-myc, Mcl-1, and ribosomal gene loci in MOLP8 MM and K562 leukemia cells, when compared with lymphoblastoid cells. Histone and DNA methylation patterns in MOLP8 cells were partially modified by the clinically promising HDAC inhibitor, vorinostat. In comparison with lymphoblastoid WIL2NS cells, MOLP8 cells and K562 cells were characterized by an absence of the gene silencing marker H3K9me2 in the c-myc and ribosomal genes. However, high levels of H3K27me3 were detected in the promoters and coding regions of selected genomic regions in these cells. Treatment by vorinostat increased the level of DNA methylation at the c-myc promoter, and this alteration was accompanied by a decrease in c-MYC protein. In MOLP8 cells, vorinostat significantly increased the H3K9 acetylation in the Mcl-1 coding regions and promoter. Both MOLP8 and K562 leukemia cells were characterized by decreased levels of H3K9me2 in the Mcl-1 gene as compared with lymphoblastoid WIL2NS cells. Lower levels of H3K9me1 in the Mcl-1 promoter, however, were specific for MM cells as compared with the other cell types studied. In other MM and leukemia cell lines, COLO677, OPM2, and U937, the ribosomal genes were less prone to epigenetic heterogeneity as compared to the c-myc and Mcl-1 proto-oncogenes. Taken together, these data describe both tumor-specific and loci-specific histone signature and DNA methylation profiles.

Interference of mouse polyomavirus with the c-myc gene and its product in mouse mammary adenocarcinomas

In two tumour sublines (T.wt/BL and T.wt/Bc), established from mammary adenocarcinomas caused by mouse polyoma (Py) infection of nu/nu mice, integration of polyomavirus DNA sequences into the c-myc gene locus was mapped. A complete Py genome was found to be integrated just upstream from the c-myc gene in T.wt/BL cell line, while only a part of the early Py region coding for the early proteins was inserted in the chromosomal DNA of T.wt/Bc cells. An interference of Py sequences with the regulation of c-myc gene expression gives further significance to a Py-derived tumour system that appears to be similar to some human mammary cancers in the modifications of c-myc expression. Both cell lines were found to produce truncated large T antigen and entire middle and small T antigens. In addition, production of VP1 protein was observed in the T.wt/BL cell line. The integration of polyomavirus sequences and/or expression of viral proteins caused an elevation of c-myc expression. The level of the c-myc expression was higher in both tumour cell lines in comparison with control normal murine mammary gland (NMuMG) lines, but substantially lower than in NMuMG cells infected with polyomavirus. Possible co-operation of Py proteins with c-Myc was examined. Through GST fusion protein pull-down experiments, we evidenced, that c-Myc forms a complex with the common part of the Py early antigens in the two tumour cell lines. Co-localisation of the c-myc and LT was observed in cells overexpressing c-Myc and LT.

Diversity and somatic hypermutation of the Ig V HDJ H, VκJκ, and VλJλ Gene Segments in Lymphoma B cells: Relevance to the origin of the neoplastic B cell clone

Burkitt’s lymphoma (BL) is a malignancy of B cells characterized by chromosomal translocations involving the immunoglobulin (Ig) and c-MYC gene loci. To address the putative role of antigen in the clonal expansion of these neoplastic B cells, we analyzed the V HDJ H and V LJ L gene segments expressed by the established cell lines derived from six endemic BL and six sporadic BL. Eight BL cell lines used genes of the V H3 family, two of the V H4, and two of the V H1. Eight V L chains were κ (four members of the Vκ3, two of the Vκ1, and two of the Vκ2 subgroups) and four λ (three members of the Vλ1 and one of the Vλ3 subgroup). The V H gene utilization was stochastic ( i.e., it reflected the relative representation of the different V H gene family members in the human haploid genome). In contrast, the V L gene utilization was skewed toward the overutilization of the Vκ3 and Vλ1 gene subgroups. When compared with those of the respective germline genes, the sequences of the expressed Ig V(D)J genes displayed nucleotide differences that resulted from somatic hypermutation. In three endemic and three sporadic BL cells, nucleotide changes yielding amino acid substitutions segregated within the complementarity determining region, indicating the application of a positive pressure for replacement mutations and suggesting that these neoplastic lymphocytes underwent a process of clonal selection driven by antigen, perhaps emerging from or transitioning through germinal centers.

Evolution of Large Extrachromosomal Elements in HL-60 Cells during Culture and the Associated Phenotype Alterations

In the HL-60 sublines that were isolated after a long-term continuous culture, abnormally stained or abnormally banded regions on chromosomes replaced extrachromosomal double minutes. The c-MYC gene is amplified in these structures. We followed the c-MYC gene loci during a consecutive passage by using FISH, and have found a large extrachromosomal element (LEE) that preexisted at the earliest passage in a very small fraction of cells. No chromosomal integration of c-MYC sequences was observed in up to 225 passages. The LEEs persistently evolved during culture and were not excluded from the nucleus. In the LEE-positive cells, the spontaneous differentiation was blocked and the granulocytic differentiation that was induced by treatment with dimethyl sulfoxide was reversed by withdrawal of the drug. The c-MYC gene integration into LEEs is unlikely to lead to these phenotypes. The reversibility might be related to the reversible c-MYC down-regulation during the early phase of the drug treatment of HL-60 cells at early cell passages.

Tissue, cell type, and breast cancer stage-specific expression of a TGF-β inducible early transcription factor gene

This laboratory has previously identified a novel TGF-beta inducible early gene (TIEG) in human osteoblasts [Subramaniam et al. (1995): Nucleic Acids Res 23:4907-4912]. Using TIEG specific polyclonal antibody and immunoprecipitation methods in normal human fetal osteoblast cells (hFOB cells), we have now demonstrated that TIEG encodes a 72-kDa protein whose levels are transiently increased at as early as 2 h of TGF-beta treatment. Polarized confocal microscopic analysis of hFOB cells shows a nuclear localized TIEG protein in untreated cells under the conditions described under Methods. Interestingly, the levels of TIEG protein in the nuclei increase when the cells are treated with TGF-beta 1 for 2 h. In contrast, similar analyses of untreated human keratinocytes show a cytoplasmic localized TIEG protein that appears to be translocated to the nucleus after H2O2 treatment. Additional immunohistochemical studies have demonstrated that TIEG protein is expressed in epithelial cells of the placenta, breast, and pancreas, as well as in osteoblast cells of bone and selected other cells of the bone marrow and cerebellum with some cells showing a cytoplasmic localization and others a nuclear localization. All cells of the kidney display negative staining for this protein. Interestingly, a stage specific expression of TIEG protein is found in a dozen breast cancer biopsies, using immunohistochemistry. The cells in normal breast epithelium displays a high expression of TIEG protein, those in the in situ carcinoma display less than one-half of the levels, and those in the invasive carcinoma show a complete absence of the TIEG protein. TIEG has been localized to chromosome 8q22.2 locus, the same locus as the genes involved in osteopetrosis and acute myeloid leukemia and close to the c-myc gene locus and a locus of high polymorphism in cancer biopsies. The correlation between the levels of TIEG protein and the stage of breast cancer, its prime location in human chromosome 8q22.2, and past studies with pancreatic carcinoma, suggests that TIEG may play a role in tumor suppressor gene activities, apoptosis, or some other regulatory function of cell cycle regulation.

Tissue, cell type, and breast cancer stage‐specific expression of a TGF‐β inducible early transcription factor gene

This laboratory has previously identified a novel TGF-β inducible early gene (TIEG) in human osteoblasts [Subramaniam et al. (1995): Nucleic Acids Res 23:4907–4912]. Using TIEG specific polyclonal antibody and immunoprecipitation methods in normal human fetal osteoblast cells (hFOB cells), we have now demonstrated that TIEG encodes a 72-kDa protein whose levels are transiently increased at as early as 2 h of TGF-β treatment. Polarized confocal microscopic analysis of hFOB cells shows a nuclear localized TIEG protein in untreated cells under the conditions described under Methods. Interestingly, the levels of TIEG protein in the nuclei increase when the cells are treated with TGF-β1 for 2 h. In contrast, similar analyses of untreated human keratinocytes show a cytoplasmic localized TIEG protein that appears to be translocated to the nucleus after H2O2 treatment. Additional immunohistochemical studies have demonstrated that TIEG protein is expressed in epithelial cells of the placenta, breast, and pancreas, as well as in osteoblast cells of bone and selected other cells of the bone marrow and cerebellum with some cells showing a cytoplasmic localization and others a nuclear localization. All cells of the kidney display negative staining for this protein. Interestingly, a stage specific expression of TIEG protein is found in a dozen breast cancer biopsies, using immunohistochemistry. The cells in normal breast epithelium displays a high expression of TIEG protein, those in the in situ carcinoma display less than one-half of the levels, and those in the invasive carcinoma show a complete absence of the TIEG protein. TIEG has been localized to chromosome 8q22.2 locus, the same locus as the genes involved in osteopetrosis and acute myeloid leukemia and close to the c-myc gene locus and a locus of high polymorphism in cancer biopsies. The correlation between the levels of TIEG protein and the stage of breast cancer, its prime location in human chromosome 8q22.2, and past studies with pancreatic carcinoma, suggests that TIEG may play a role in tumor suppressor gene activities, apoptosis, or some other regulatory function of cell cycle regulation. J. Cell. Biochem. 68:226–236, 1998. © 1998 Wiley-Liss, Inc.

The absence of ongoing immunoglobulin gene hypermutation suggests a distinct mechanism for c-myc mutation in endemic Burkitt's lymphoma.

Burkitt's lymphoma is a malignancy of mature, immunoglobulin (Ig)-bearing B cells characterized by translocation between c-myc and Ig gene loci. A role for the juxtaposed Ig genes in the mutation and deregulation of c-myc expression typical of endemic Burkitt's lymphoma (eBL) has been proposed, but never proven. Our objective was to determine whether Ig gene hypermutation is ongoing in eBL. We isolated Ig heavy-chain sequences from K962 eBL tumor cells using reverse transcription and polymerase chain reaction (PCR) amplification. The PCR product was ligated into Bluescript II vectors. Multiple subclones were sequenced and the variable regions were compared for evidence of ongoing Ig hypermutation. Six total single base substitutions were observed within four of the nine subclones studied. Four substitutions resulted in amino acid changes and two were silent. There was no clustering of mutations in hypervariable regions, or a high incidence of amino acid replacement or link substitutions, all of which are characteristic of Ig hypermutation. The observed mutations occurred at a rate consistent with Taq polymerase error. Our data indicate that in the eBL tumor sample K962, the mechanism underlying c-myc mutation is distinct from that which gives rise to Ig hypermutation.

Long-range Fragmentation of the Eukaryotic Genome by Exogenous and Endogenous Nucleases Proceeds in a Specific Fashion via Preferential DNA Cleavage at Matrix Attachment Sites

Small cell lung cancer cells (OC-NYH-VM) were permeabilized and treated with different nucleases. The long-range distribution of DNA cleavage sites in the amplified c-myc gene locus was then analyzed by pulsed field gel electrophoretic separation of the released 50-kilobase to 1-megabase DNA fragments followed by indirect end labeling. Exogenous DNase I and nucleases specific for the single-stranded DNA were found to generate similar nonrandom patterns of large DNA fragments. The cleavage sites were located close to or even colocalized with matrix attachment regions, which were mapped independently using a recently developed procedure for DNA loop excision by DNA topoisomerase II-mediated DNA cleavage. Endogenous acidic nuclease with the properties of DNase II also digested DNA preferentially in proximity to the matrix attachment regions, generating characteristic patterns of excised DNA loops and their oligomers. A similar, although less specific, pattern of DNA fragmentation was observed after incubation of permeabilized cells under conditions favoring the activity of endogenous neutral Ca(2+)- and Mg(2+)-dependent nucleases. These findings are discussed in the context of the current model of the spatial domain organization of eukaryotic genome.

The Functional Basis of c-myc and bcl-2 Complementation during Multistep Lymphomagenesis in Vivo

Oncogenes are known to be deregulated by chromosomal translocations occurring at high frequency in specific malignancies. Among the most well characterized of these are c-myc, associated with the t(8;14) in Burkitt's lymphomas, and bcl-2, associated with the t(14;18) in follicular lymphomas. In addition to their role in regulating rates of proliferation, it is known that oncogenes and tumor suppressor genes can also regulate rates of apoptotic cell death. The contribution of c-myc and bcl -2 to the regulation of cell death during lymphomagenesis in vivo is assessed using bcl-2-Ig and Eμ-myc transgenic mice and bcl-2/myc hybrid transgenic mice. Translocations between the endogenous c-myc gene and immunoglobulin loci, e.g., t(12;15), are common in lymphomas arising in the bcl-2-Ig mice. Furthermore, bcl-2/c-myc double transgenic mice exhibit accelerated lymphomagenesis, indicating cooperation between these two oncogenes. Genetic complementation of c-myc and bcl-2 during lymphomagenesis resulted from the suppression of c-myc-associated apoptosis. Other genes are likely involved in regulating cell death during multistep lymphomagenesis.

Different topoisomerase II antitumor drugs direct similar specific long-range fragmentation of an amplified c-MYC gene locus in living cells and in high-salt-extracted nuclei.

We have analyzed the long-range distribution of topoisomerase II-mediated cleavages induced in an amplified human c-MYC gene locus in the presence of several antitumor agents. The long-range cleavage patterns were found to be nonrandom and similar for all antitumor drugs tested. Cleavages occurred within several kilobase-long areas (approximately 5 kb) highly accessible to topoisomerase II and separated by extended regions (approximately 70-100 kb) of less accessibility, possibly reflecting the mode of DNA organization into loops along the chromosome. Within the cleavage areas, the patterns of cleavage sites showed a certain dependence on the type of drug used for entrapment of topoisomerase II-DNA complexes. Importantly, distribution of cleavage areas in native chromatin and histone-depleted nuclei was very similar, if not identical, suggesting that the primary target of antitumor agents in vivo is topoisomerase II associated with the high-salt-insoluble nuclear matrix. These data show that matrix-attached DNA is preferentially damaged by topoisomerase II-targeting agents, which may be an important cellular event contributing to drug-induced cell death.

Changes in promoter utilization in human and mouse c-myc genes upon transformation induction in temperature-sensitive cell lines.

We have previously reported accelerated transcription and rapid accumulation of c-myc mRNAs upon induction of transformation in a temperature-sensitive mouse cell line (Gallant et al., 1989, Oncogene Res., 4:39-46). Here we have used both mouse and human cell lines transformed with a temperature-sensitive mutant of the Simian virus 40 (SV40) virus to investigate whether a shift in promoter utilization within the c-myc gene locus is part of a general mechanism that deregulates c-myc expression during transformation induction. We devised a simple and sensitive method using reverse transcription followed by radioactive polymerase chain reaction (RT-PCR) to measure the relative change in c-myc mRNAs arising from each of the four known promoters. We show that a three to fivefold increase in c-myc transcripts from the P1 and P3 promoters occurs in both human and mouse cell lines within 30 min of the shift to the permissive temperature. The major P2-initiated transcripts are not significantly effected. However, exon 3-containing RNAs increase more gradually up to 24 h postinduction and P1 and P3 transcripts, while remaining elevated, still contribute relatively little to the total c-myc RNA population. These and other results, demonstrating a transient activation of P1 and P3 promoters, suggest an indirect role of the minor transcripts in the deregulated expression of the c-myc gene in transformed cells.

Cytogenetic Rearrangement of C-MYC Oncogene Occurs Prior to Infection with Epstein-Barr Virus in the Monoclonal Malignant B Cells From an AIDS Patient

Two cell lines were originated from the peripheral blood (PB-LAM) and bone-marrow (BM-LAM) of a patient with Burkitt-type acute lymphoblastic leukemia and AIDS. 26 and 7 clones were isolated from PB-LAM and BM-LAM respectively by limiting dilution. All of these had surface IgM lambda and the CD10 marker with low to absent CD23, CD30, CD39 and surface adhesion molecules. Furthermore, they shared the same chromosomal abnormalities (trisomy 7 and t(8;14) translocation) and the same rearrangements of immunoglobulin L and H chain and of c-myc gene loci. These features are those most frequently found in Burkitt's lymphoma (BL) cells and were different from those of the parental cell lines, which, besides cells identical to those of the malignant clones, also contained normal lymphoblastoid cells. Therefore, the cloning procedure used selected for the growth of cells with malignant features. EBV latent antigens were detected in all clones by Western blotting and their pattern of expression resembled that usually observed in BL cells. All the clones were positive for the EBV genome by Southern blotting and had monomorphic EBV-fused termini as determined by using cDNA probes specific for sequences at either end of the viral genome. However, the clones derived from PB-LAM had EBV fused termini of a different size from that of the clones derived from BM-LAM. The presence of different EBV-fused termini in otherwise monoclonal malignant cells indicate that EBV infection was possibly a late event in lymphomagenesis following rearrangement of the c-myc and the Ig gene loci.

Insertion of SMRV-H viral DNA at the c-myc gene locus of a BL cell line and presence in established cell lines.

Mutation of the c-myc gene locus has occurred during in vitro culture of the Burkitt lymphoma cell line Namalwa. Gene cloning and sequencing of this c-myc gene locus has revealed the insertion of incomplete copies of the Squirrel Monkey Retrovirus-Human (SMRV-H) proviral genome. Insertions of the SMRV-H genome were seen in all Namalwa cell lines tested, with multiple insertions being seen in some sublines. Viral particles with the morphology of type-D retrovirus, budding from these cells, were seen by transmission EM. New insertions of the genome were produced spontaneously in these cells. The origins of the type-D retrovirus and its implications for workers in the field are discussed.

PCS, a gene related to the immunoglobulin super family of axonal glycoproteins is expressed in murine plasma cell tumors.

Intraperitoneal plasma cell tumors (PCTs) are induced by pristane (tetramethylpentadecane), a branched-chain alkane paraffin oil, in BALB/cAn and other susceptible mouse strains with minimal to mean latencies of 120 to 210 days respectively (Potter, 1972). PCTs harbor one of two reciprocal chromosome translocations always involving band D2/3 on chromosome 15 and either the Immunoglobulin heavy chain (IgH) locus on 12 or the kappa light chain locus (IgK) on 6 (Ohno et al., 1979). Band D2/3 was subsequently found to be the c-myc gene locus and the activation of this proto-oncogene by such chromosomal rearrangements has been shown to be a contributing factor for plasmacytomagenesis (reviewed by Mushinski, 1988; Marcu et al., 1992). Plasmacytomagenesis was rapidly accelerated (50–120 day mean latencies) upon injection of oncogene-containing retroviruses (v-abl, myc and raf or myc and ras) along with pristane (Potter et al., 1973; Ohno et al., 1984; Potter et al., 1987; Clynes et al., 1988; Troppmair et al., 1989) and viruses-containing an activated myc oncogene induced PCTs without c-myc associated chromosome translocations (Potter et al., 1987; Clynes et al., 1988; Troppmair et al., 1989). Recently, a retrovirus harboring v-abl and c-myc was found to be the most potent oncogene combination inducing PCTs in 100% of adult BALB/c mice with or without a wild type Moloney helper virus and also in the absence of pristane (Weissinger et al., 1991). These observations would collectively argue that multiple genetic events in addition to c-myc activation are required for malignant plasma cell tumor formation. Indeed, at least three recessive genetic loci are believed to confer resistance to PCT formation in non-sensitive mouse strains but the identities of these genes and other dominant acting ones in addition to myc remain unknown.