Lack of mutagenicity and clastogenicity of PNAEmu-NLS targeted to a regulatory sequence of the translocated c-myc oncogene in Burkitt's lymphoma

Peptide nucleic acids (PNAs) are synthetic homolog of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible polyamide. They bind complementary polynucleotide sequences with higher affinity and specificity than their natural counterparts. PNAs linked to the appropriate nuclear localization signal (NLS) peptide have been used to selectively down-regulate the expression of several genes in viable cells. For example in Burkitt's lymphoma (BL) cells the c-myc oncogene is translocated in proximity to the Emu enhancer of the Ig gene locus and upregulated. PNAs complementary to the second exon of c-myc or to the Emu enhancer sequence (PNAEmu-NLS), selectively and specifically block the expression of the c-myc oncogene and inhibit cell growth in vitro and in vivo. PNAEmu-NLS administration to mice did not exhibit toxic effects even at the highest concentration allowed by the experimental conditions. Because of the accumulating data confirming PNAEmu-NLS potential therapeutic value, PNAEmu-NLS was evaluated for the inability to induce mutations in tester strains of Salmonella typhimurium, Escherichia coli, and at the hprt locus in Chinese hamster ovary cells (CHO). Moreover, the induction of chromosomal aberrations in CHO cells and of micronuclei in human lymphocytes were investigated. We may conclude that PNAEmu-NLS neither induces mutations nor has clastogenic effects as detectable by treatment under the standard test conditions.     

Aberrant c-myc RNAs of Burkitt's lymphoma cells have longer half-lives.

BL67 and BL18 are Burkitt's lymphoma cell lines with t(8;14) translocations (the breakpoint is in the first exon and first intron, respectively) in which the mu-heavy chain switch region is fused to the c-myc gene in head to head orientation. In both cell lines only aberrant c-myc RNAs are found. BL67 cells contain two c-myc RNA species of 2.4 and 3.5 kb. The 2.4-kb RNA is initiated at several cryptic promoters in the first intron. The 3.5-kb RNA is transcribed from the immunoglobulin heavy chain anti-sense strand across the breakpoint of the translocation into the first exon of the c-myc gene and is then normally spliced using the physiological splice donor and acceptor sites of the c-myc gene. BL18 contains c-myc RNA of 2.4 kb initiated at cryptic promoters in the first intron and additional RNAs of 0.90 kb and 0.74 kb transcribed from the dual c-myc promoters on the reciprocal fragment of the translocation. The cytoplasmic turnover of these RNAs differs significantly from that of the normal c-myc message. The 3.5-kb RNA of BL67 cells and the 0.90-kb and 0.74-kb RNAs of BL18 cells, which are both hybrid molecules consisting of c-myc and immunoglobulin sequences, have a half-life of several hours in contrast to the normal c-myc message with a half-life of 15 min. The aberrant 2.4-kb c-myc RNAs of BL67 and BL18 cells are also more stable than the normal c-myc message and disappear with a half-life of 50 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free radicals act as effectors in the growth inhibition and apoptosis of iron-treated Burkitt's lymphoma cells

The addition of ferric citrate to Burkitt's lymphoma (BL) cell lines inhibits growth, leads to the accumulation of cells in the phase G₂/M of the cell cycle and to the modulation of translocated c-myc expression. The increase in the labile iron pool (LIP) of iron-treated BL cells leads to cytotoxicity. Indeed, intracellular free iron catalyzes the formation of highly reactive compounds such as hydroxyl radicals and nitric oxide (NO) that damages macromolecular components of cells, eventually resulting in apoptosis. In this report, we have investigated the possible involvement of free radicals in the response of Ramos cells to iron. When added to Ramos cells, iron increased the intracellular levels of peroxide/peroxynitrite and NO. Moreover, the addition of free radicals scavengers (TROLOX^® and Carboxy-PTIO) neutralized the effects of iron on Ramos cells while addition of an NO donor or hydrogen peroxide (H₂O₂) to cells generated effects which partially mimicked those induced by iron addition. Collectively, our results suggest the involvement of free radicals as effectors in the iron specific growth inhibition of BL cells observed in vitro.     

The peptide nucleic acid targeted to a regulatory sequence of the translocated c-myc oncogene in Burkitt's lymphoma lacks immunogenicity: follow-up characterization of PNAEmu-NLS

The present study aims to evaluate the antigenicity of a PNA complementary to the Emu sequence (PNAEmu) with cancer therapeutic potential properties in Burkitt's lymphoma (BL). In BL cells, the c-myc oncogene is repositioned next to the Emu enhancer of the immunoglobulin (Ig) locus, due to chromosomal translocation, and up-regulated. PNAEmu linked to a nuclear localization signal peptide was shown specifically to block c-myc hyperexpression by inhibiting cell growth in vitro and in vivo. Recently, we reported that the administration of PNAEmu to mice, following inoculation with BL cells, hinders tumor growth without toxic effects. To investigate the potential use of PNAEmu in clinical applications further, we tested its antigenicity. Mice were inoculated with an emulsion of free PNA or PNA crosslinked to the immunogenic carrier keyhole limpet hemocyanin (KLH) with Freund's adjuvant. Antibodies to free PNA were undetected, whereas both IgG and IgM antibodies to PNA-KLH were detected in mouse serum 28 and 38 days after inoculation.     

Free radicals act as effectors in the growth inhibition and apoptosis of iron-treated Burkitt's lymphoma cells

The addition of ferric citrate to Burkitt's lymphoma (BL) cell lines inhibits growth, leads to the accumulation of cells in the phase G₂/M of the cell cycle and to the modulation of translocated c-myc expression. The increase in the labile iron pool (LIP) of iron-treated BL cells leads to cytotoxicity. Indeed, intracellular free iron catalyzes the formation of highly reactive compounds such as hydroxyl radicals and nitric oxide (NO) that damages macromolecular components of cells, eventually resulting in apoptosis. In this report, we have investigated the possible involvement of free radicals in the response of Ramos cells to iron. When added to Ramos cells, iron increased the intracellular levels of peroxide/peroxynitrite and NO. Moreover, the addition of free radicals scavengers (TROLOX^® and Carboxy-PTIO) neutralized the effects of iron on Ramos cells while addition of an NO donor or hydrogen peroxide (H₂O₂) to cells generated effects which partially mimicked those induced by iron addition. Collectively, our results suggest the involvement of free radicals as effectors in the iron specific growth inhibition of BL cells observed in vitro.     

Amplification of the translocated c-myc genes in three Burkitt lymphoma cell lines

Translocations of the coding exons of the human c-myc gene are consistent features of human Burkitt lymphomas (BL). In the BL cell lines CA46, JD40, and ST486, the second and third c-myc exons have been translocated into the immunoglobulin heavy chain locus. In addition to this rearrangement, in all three cell lines, we have found that the translocated c-myc exons show low-level amplification relative to restriction fragments from the germ-line c-myc gene. The patterns of hybridization of an IgM switch region probe suggest that immunoglobulin heavy chain sequences have been co-amplified with the translocated c-myc sequences. Differential sedimentation was used to determine whether the amplified sequences reside in high-molecular-weight chromosomes or low-molecular-weight extrachromosomal DNA. In JD40 and ST486 cells, the amplified c-myc sequences were found on high-molecular-weight chromosomes; ST486 cells also contained translocated c-myc sequences in low-molecular-weight, extrachromosomal DNA, as did CA46 cells. These conclusions were corroborated by fluorescence in-situ hybridization (FISH) of HeLa, CA46, ST486 and JD40 metaphase chromosomes. These results suggest that there is ongoing selection for cells containing amplified copies of the expressed c-myc sequences, and that there is continuous generation of extrachromosomal copies of the translocated c-myc sequences in ST486 and CA46 cells.     

Expression of p53, bcl-2, bax, bcl-x2 and c-myc in radiation-induced apoptosis in Burkitt's lymphoma cells

Apoptosis, a form of physiological cell death, is a genetically determined program essential for normal development and maintenance of tissues, which has been linked to a variety of gene products. We have examined the susceptibility to radiation-induced apoptosis of cell lines derived from the human B cell tumour, Burkitt's lymphoma (BL), displaying a variety of phenotypic characteristics and expressing genes implicated in apoptosis at different levels. The susceptibility to apoptosis following gamma radiation varied significantly amongst the lines. Cell lines with wild type p53 were susceptible to radiation-induced apoptosis but two of five BL lines with only mutant p53 allele also displayed similar susceptibility. Some BL cell lines that expressed bcl-2 at levels comparable with Epstein-Barr virus (EBV) transformed normal B cells were highly susceptible to gamma radiation-induced apoptosis, whereas others expressing low levels were resistant. When these lines were analysed for bax and bcl-X(L) expression again no correlation was observed with susceptibility or resistance to apoptosis. Two BL cell lines having deregulated expression of c-myc were resistant to the induction of apoptosis while two others which had regulated c-myc expression were susceptible. Thus the status of p53, c-myc, bcl-2, bcl-X(L) and bax is not sufficiently informative in BL lines to predict susceptibility to radiation-induced apoptosis.     

Expression of normal and translocated c-myc alleles in Burkitt''s lymphoma cells: evidence for different regulation.

In Burkitt's lymphoma (BL) cells the normal c-myc allele is usually silent or expressed at very low levels. Here we demonstrate that the normal c-myc allele can be induced in BL cells by 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA did activate the normal c-myc alleles in Raji(P207), BL36, P3HR1, Jijoye and LY91 cells, but not in Raji(DE88), BL41, BL67, LY47 and KK124 cells. C-myc RNA derived from the normal allele appeared 6 h after treatment with TPA and showed the characteristic preferential usage of the second promoter. This induction could not be inhibited by cycloheximide. Despite the differences in c-myc induction in Raji(P207) and Raji(DE88) cells, c-fos and the early Epstein-Barr virus gene DR were induced to a similar extent and with similar kinetics by TPA. Nuclear run-on experiments suggest that the normal c-myc allele in Raji cells is activated at least in part by releasing a block to RNA elongation at the end of c-myc exon 1. Expression of the translocated c-myc alleles was also affected by TPA; however, only if cycloheximide was simultaneously present. TPA plus cycloheximide induced a rapid decrease of c-myc RNA derived from the translocated allele within 6 h, whereas cycloheximide alone led to abolition of c-myc RNA after 16-24 h. This rapid decline of c-myc RNA was observed in Raji and BL41 cells, but not in three cell lines with variant t(2;8) and t(8;22) translocations.     

NF-kappa B sites function as positive regulators of expression of the translocated c-myc allele in Burkitt''s lymphoma.

An in vivo footprint over a potential NF-kappa B site in the first exon of the c-myc gene has been identified on the translocated allele in the Ramos Burkitt's lymphoma cell line. The potential NF-kappa B site in the 5' flanking sequence of c-myc was found to be occupied on the translocated allele in the Raji Burkitt's cell line. Electrophoretic mobility shift assays with each of these sequences demonstrated complexes with mobilities identical to those of the NF-kappa B site from the kappa light-chain gene. A supershift was obtained with anti-p50 antibody with the exon site. The upstream-site shift complex disappeared with the addition of anti-p50 antibody. Binding of NF-kappa B proteins to the c-myc exon and upstream sites was demonstrated by induction of binding upon differentiation of pre-B 70Z/3 cells to B cells. UV cross-linking experiments revealed that a protein with a molecular mass of 50 kDa bound to the exon and upstream sites. Transfection experiments with Raji cells demonstrated that both sites functioned as positive regulatory regions, with a drop in activity level when either site was mutated. Access to these sites is blocked in the silent normal c-myc allele in Burkitt's lymphoma cells, while Rel family proteins bind to these sites in the translocated allele. We conclude that the two NF-kappa B sites function as positive regulatory regions for the translocated c-myc gene in Burkitt's lymphoma.     

The decrease in polyamine levels is not associated with growth inhibition in suspension-cultured rice cells under nitrogen deficiency

The effects of nitrogen deficiency on the growth and polyamine levels of suspensioncultured rice cells were investigated. Nitrogen deficiency markedly inhibited cell growth and resulted in lower levels of putrescine, spermidine and spermine than in the control culture supplied with nitrogen. The growth inhibition of rice cells induced by nitrogen deficiency could be recovered by the addition of a mixture of amino acids (glycine, L-aspartic acid, L-glutamic acid and L-arginine), but not by polyamines (putrescine, spermidine or spermine). Our results indicate that the decline of polyamine levels is not a factor causing growth inhibition of suspension-cultured rice cells under nitrogen deficiency.     

Phorbol ester-induced inhibition of proliferation of Daudi Burkitt's lymphoma cells by impairment of cytokinesis

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) exerts a dose-dependent effect on Daudi cell proliferation. A low concentration has a slight mitogenic effect but higher concentrations inhibit proliferation. The inhibitory effect is associated with increases in cell size, macromolecular content, and incorporation of precursors into RNA and protein. Cell cycle analysis indicates that TPA at 1–10 nM leads to an apparent accumulation of cells in G₂/M phase. However, within this population a significant proportion of cells undergo nuclear division but fail to carry out cytokinesis, giving rise to cells with two or more nuclei. Consistent with this, DNA synthesis continues in cells which cease to divide in the presence of TPA. The ability of the phorbol ester to inhibit proliferation can thus be attributed mainly to an inhibition of cytokinesis rather than DNA replication     

Regulation of expression of c-fos and c-myc in rat lymphoma Nb-2 cells

This study examined the effects of the mitogen, prolactin and the cell cycle inhibitors, cyclosporin A and neomycin sulfate, on expression of the proto-oncogenes c-fos and c-myc in the rat lymphoma Nb-2 cell line. Stimulation of quiescent cultures with prolactin resulted in a 2-3-fold increase in the constitutive levels of c-myc mRNA which peaked at 4 h and declined thereafter. c-Fos mRNA was not detected in quiescent or prolactin-stimulated cultures. Cyclosporin A or neomycin sulfate reversibly blocked the mitogenic effect of prolactin on Nb-2 cells, but had little effect on constitutive levels of c-myc. However, the release of Nb-2 cells from a cyclosporin A or a neomycin sulfate block resulted in a rapid transient induction of c-fos which peaked at 0.5-1 h and declined rapidly thereafter. These results indicate that the rapid transient expression of c-fos following release from cell cycle blockage was not sufficient to elicit cell division, but these cells were competent to respond to prolactin. Prolactin allows progression through the cell cycle and enhances c-myc mRNA levels.     

Effect of nucleolin on adriamycin resistance via the regulation of B-cell lymphoma 2 expression in Burkitt's lymphoma cells

Nucleolin (NCL, C23) is an important nucleocytoplasmic multifunctional protein. Due to its multifaceted profile and high expression in cancer, NCL is considered to be a marker of drug resistance associated with chemotherapy. However, the biochemical mechanisms in which NCL suppresses drug sensitivity in several cancers have yet to be fully elucidated. This study aims to explore the effect of NCL on drug sensitivity and its potential mechanism in CA46 Burkitt's lymphoma (BL) cells. CA46 BL cells were transfected with lentiviruses carrying the NCL gene (CA46-NCL-overexpression, CA46-NCL-OE), or shRNA sequences that target the endogenous NCL gene (CA46-NCL-knockdown, CA46-NCL-KD). Adriamycin (ADM) IC50 levels for CA46-NCL-overexpressed (OE), CA46-NCL-OE control (OEC), CA46-NCL-knockdown (KD), and CA46-NCL-KD control (KDC) cells were 0.68 ± 0.06 μg/ml, 0.68 ± 0.06 μg/ml, 0.68 ± 0.06 μg/ml, and 0.30 ± 0.04 μg/ml, respectively. Apoptosis rates were significantly increased following NCL KD, whereas the opposite effect was noted in OE cells. A significant reduction of B-cell lymphoma 2 (Bcl-2) mRNA and protein levels in KD cells was observed, while OE cells displayed the opposite effect. The stability of Bcl-2 mRNA was influenced by NCL levels, the half-life of which was extended after NCL-OE, whereas it was reduced in KD cells. Finally, results of RNA-immunoprecipitation assays indicated that NCL could bind to Bcl-2 mRNA in CA46 cells. Taken together, these results suggested that NCL could mediate Bcl-2 expression and stability, and thus enhance ADM resistance in CA46 BL cells.