Circular antisense oligonucleotides inhibit growth of chronic myeloid leukemia cells.

BACKGROUND: Antisense represents a conceptually powerful method for regulating gene expression. However, antisense oligonucleotides developed to date manifest two serious limitations-nuclease susceptibility and nonspecific hybridization. Circular oligonucleotides may be superior to conventional linear oligonucleotides in both respects. First, circular agents, having no ends, are exonuclease-resistant. Second, they bind to complementary strands of RNA and DNA with a higher affinity than corresponding linear agents. METHODS AND RESULTS: We assessed the activity of circular phosphodiester deoxynucleotides using chronic myeloid cell lines by targeting polypurine sequences. To represent cells having a bcr3/abl2-type junction, we used K562 cells. A circle targeting a bcr polypurine sequence 385 nucleotides 5' to the junction decreased the cell number by day 5 with an IC(50) of 9 microM. To represent cells having a bcr2/abl2-type junction, we used BV173 cells. A circle targeting the bcr-abl junction itself decreased the cell number by day 7 with an IC(50) of 8 microM. Control oligonucleotides, whether the same sequence uncircularized or circles with the same nucleotide composition but in scrambled sequence, had little effect. Unlike linear agents, circles were stable when incubated in 10% serum. The amount of bcr-abl protein detected by Western blotting using a specific anti-bcr-abl antibody at 24 hr in antisense-treated BV173 cells was only 10% of that of cells treated with control circles, which demonstrates an antisense mechanism of action. CONCLUSIONS: Circular oligodeoxyribonucleotides (1) inhibit the accumulation of CML cells, (2) decrease the amount of bcr-abl protein per cell, (3) have sequence-selective activity, and (4) are more active than linear oligonucleotides containing only the base-pairing region.

Telomere-related components are coordinately synthesized during human T-lymphocyte activation.

Since telomerase activity is present in most malignant cells, but absent in most normal cells, its induction in normal cells warrants scrutiny. Therefore we have analyzed the inducibility of telomere-related components in normal lymphocytes during their activation. Telomerase activity increased over 400-fold, telomerase reverse transcriptase (hTERT) mRNA 52 x , telomerase RNA 32 x , TTAGGG repeat binding factor 1 mRNA 19 x , TTAGGG repeat binding factor 2 mRNA 20 x , and telomerase-associated protein mRNA 17 x . The peak value for each was reached at about 72 h. However hTERT rose fastest and synchronously with telomerase activity. Thus in normal human lymphocytes (1) the syntheses of all cloned telomerase-related components are coordinately regulated and (2) hTERT may have a priming role.

Human lymphocyte telomerase is genetically regulated.

Research on both cancer and aging has focused attention on the regulation of telomerase, the enzyme that synthesizes the ends of chromosomal DNA. To analyze the relative importance of genetic vs. environmental factors in determining telomerase inducibility, we have compared telomerase activity in phytohemagglutinin-stimulated peripheral blood lymphocytes from monozygotic and dizygotic twin pairs. The heritability calculated was 0.814, indicating that lymphocyte inducible telomerase activity is determined principally by genetic rather than by environmental factors.

The effect of bcr-abl antisense oligonucleotide on DNA synthesis and apoptosis in K562 chronic myeloid leukemia cells.

Mutations in oncogenes have traditionally been viewed as inducing malignancy by causing excessive cell division. However, an additional possible tumorigenic mechanism is inhibition of normally occurring apoptosis. We have studied the mechanism of action of bcr-abl in chronic myeloid leukemia (CML) by inhibiting its expression using antisense oligonucleotides. K562 cells, derived initially from a patient with CML, were incubated with 16 microM 3',5'-capped bcr-abl antisense phosphodiester 18mer targeting the bcr-abl junctional sequence. Antisense reduced cell number by day 5 by 44% +/- 2.5% S.E. compared to nonsense or no-oligomer controls. Compared to nonsense oligomer, antisense oligomer reduced [3H]thymidine incorporation by only 13% +/- 1%. By the more reliable bromodeoxyuridine incorporation method, antisense had no inhibiting effect on DNA synthesis. In contrast to its minimal effect on DNA synthesis, antisense had a large effect on apoptosis. At day 4, after 3 days of oligomer treatment, antisense increased the proportion of cells with less than 2 N DNA 2.5 +/- 0.3-fold compared to nonsense, as revealed by analysis of DNA distribution following propidium iodide-staining. After 3 days of oligomer treatment and 24 h of serum deprivation, antisense increased the proportion of cells with less than 2 N DNA even more, over 3.1 +/- 1.1-fold compared to nonsense. Because CML cells are resistant to the induction of apoptosis (as judged by DNA laddering on electrophoresis, which requires double-stranded breaks), we also assayed the binding of terminal deoxynucleotidyl transferase (TdT), which requires only single-stranded DNA breaks. Antisense treatment for 3 days increased TdT binding at day 4 by 16.4 +/- 8.7-fold. We conclude that, in CML, bcr-abl may lead to the accumulation of myeloid cells to a greater extent by inhibiting apoptosis than by increasing cell division. This bcr-abl induced inhibition of apoptosis may thwart chemotherapy and foster the accumulation of further mutations leading to the development of the blastic phase of the disease.

The neurofibroma in von Recklinghausen neurofibromatosis has a unicellular origin.

von Recklinghausen neurofibromatosis (NF1) is the most common hereditary syndrome predisposing to neoplasia. NF1 is an autosomal dominant disease caused by a single gene which maps to chromosome 17q11.2. The most common symptomatic manifestation of NF1 is the benign neurofibroma. Our previous studies of tumors in NF1, studies which detected a loss of heterozygosity for DNA markers from the NF1 region of chromosome 17 in malignant tumors, did not detect a loss in neurofibromas. We report here that a more extensive study, including the analysis of neurofibromas from 19 unrelated NF1 patients by using seven probes, failed to detect a single instance of loss of heterozygosity. This finding suggests that neurofibromas are either polyclonal or monoclonal in origin but arise by a mechanism different from that of NF1 malignancies. In order to investigate the first possibility, we analyzed neurofibromas from female NF1 patients by using an X chromosome-specific probe, from the phosphoglycerokinase (PGK) gene, which detects an RFLP. The detected alleles carry additional recognition sites for the methylation-sensitive enzyme HpaII, so that the allele derived from the active X chromosome is digested by HpaII while the one from the hypermethylated, inactive X chromosome is not. We analyzed neurofibromas from 30 unrelated females with NF1. Eight patients were heterozygous for the PGK RFLP. By this assay, neurofibromas from all eight appeared monoclonal in origin. These results suggest that benign neurofibromas in NF1 arise by a mechanism that is different from that of malignant tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular genetic analysis of tumors in von Recklinghausen neurofibromatosis: loss of heterozygosity for chromosome 17.

The most common inherited syndrome in man predisposing to neoplasia is neurofibromatosis-1 (von Recklinghausen disease) (NF1). We investigated the hypothesis that affected individuals carry a single inactive allele at the NF1 locus in the germline and that a tumor arises from a cell in a susceptible tissue in which the remaining normal allele has been lost or inactivated. DNA from tumor and nontumor tissue from 27 NF1 patients was analyzed with three markers closely linked to the NF1 locus and two additional markers from chromosome 17. No loss of heterozygosity was observed in neurofibromas, plexiform or not. For other tumor types analyzed, seven of 14 showed a loss. A loss of heterozygosity was observed in six of 11 of the malignant peripheral nerve tumors analyzed. Of the seven malignancies demonstrating a loss, five involved a neurofibrosarcoma. These findings suggest that the pathogenesis of neurofibrosarcoma in NF1 involves a deficiency of the NF1 gene product. In any given patient, loss of heterozygosity was detected at some marker loci but not others. Thus the mutations demonstrated in these tumors comprise a set of overlapping mutations, which may facilitate more precise localization of the NF1 gene.

Induction of megakaryocytic characteristics in human leukemic cell line K562: polyploidy, inducers, and secretion of mitogenic activity.

Because of the rarity of megakaryocytes in the bone marrow, a cell line inducible for megakaryocytic characteristics provides a valuable model for study. When cultured with phorbol esters, the human multipotent hematopoietic leukemic cell line K562 can be induced to develop many megakaryocytic characteristics, viz. increased cell size, reduced growth rate, megakaryocytic antigens, and expression of the sis proto-oncogene, the structural gene for the B-chain of platelet-derived growth factor. Further aspects of this process are here presented. First, it induces the release of mitogenic activity into the medium. Second, phorbol dibutyrate induces polyploidy, a feature of normal megakaryocyte development. Third, mezerein and teleocidin, nonphorbol ester tumor promotors, also induce development of multinuclearity and polyploidy.

K562 cell erythroid differentiation: requirement for a factor in fetal bovine serum.

K562 human erythroleukemia cells can be induced to make hemoglobin by a variety of inducing agents. Most of these agents are effective in media supplemented with fetal bovine serum (FBS), but not in media supplemented with newborn bovine serum (NBS). The active factor in FBS has an apparent molecular weight of 30,000 daltons and appears to be a protein on the basis of the following properties: lability at 100 degrees C, inactivation by desferrioxamine plus trypsin, resistance to periodate, and resistance to ribonuclease. Media containing NBS can be used for induction if supplemented by either this factor or transferrin of bovine or human origin. The small size of the active factor (mol. wt. approximately 30,000 daltons) indicates that it is not identical to bovine transferrin (mol. wt. approximately 77,000 daltons). However, when iron-saturated bovine transferrin is digested with trypsin, the peptide fragments produced resemble the FBS factor in activity, size, and reaction with antibovine serum transferrin.

Acquired hemoglobin H disease in idiopathic myelofibrosis.

A 68-year-old male, diagnosed 1 year previously as having myelofibrosis, developed hemolysis, red cell inclusions, and 37% Hb H. The alpha/beta globin synthetic ratio for circulating reticulocytes, determined by 3H-leucine incorporation and globin chain separation by carboxymethylcellulose chromatography in urea, was 0.049. When total RNA was purified from peripheral blood cells and translated in a wheat germ cell-free translation system, the alpha/beta ratio of the translation products was 0.26, indicating mRNA as a major cause of the globin synthetic imbalance. This study demonstrates that myelofibrosis is one setting in which acquired Hb H disease occurs; that the synthetic imbalance may be extreme; and that it can be associated with an imbalance in the activities of specific globin mRNAs.