Oligonucleotides can specifically target not only nucleic acids but also proteins. Some proteins recognizing oligonucleotides in a sequence-specific manner have been related to cancer transformation and progression. We have found that oligonucleotides composed by repeated and/or variable intervals of GT n with 1 р n р 7, are able to exert a specific and dose-dependent growth inhibition on human CCRF-CEM, CEM-VLB300, U937, Jurkat, H9 and HeLa tumor cell lines. In contrast, G→C, G→A, T→C and T→A base substituted control oligonucleotides do not significantly alter cellular growth. In all cell lines, a nuclear protein (molecular mass ϭ 45Ϯ7 kDa), which specifically recognizes GT n, was identified. Our hypothesis is that the formation of the GT n -protein complex in human cancer cell lines may be involved in the growth inhibition effect. In fact, we found that the reduction or lack of cytotoxic effects by GT n in phorbol 12-myristate 13-acetate-treated CCRF-CEM cells and in normal human lymphocytes is paralleled by the simultaneous reduction or lack of GT n -protein complex. Oligonucleotides specifically 'quenching' intracellular protein activities by forming oligonucleotide-protein complexes may be of potential interest in the treatment of human tumors.

Background:We measured anti-transglutaminase (antitTG) antibody in the culture medium of intestinal biopsy specimens from patients with suspected celiac disease (CD) and evaluated the relationship between antibody production and severity of intestinal mucosal damage. Methods: We performed diagnostic testing for CD on 273 consecutive patients. In addition to routine histologic evaluation of duodenal biopsy specimens, we assayed anti-tTG antibodies in serum and in the culture medium of duodenal biopsy specimens. Results: CD was diagnosed in 191 of the 273 patients. Sensitivity and specificity of the serum anti-endomysium (EmA) and anti-tTG assays were 83% and 85% and 99% and 95%, respectively, and both had 88% diagnostic accuracy. EmA and anti-tTG assayed in the culture medium had 98% sensitivity, 100% specificity, and 98% diagnostic accuracy (vs serum assays; P <0.0001). Twenty-nine CD patient specimens (16%) were negative for serum anti-tTG and EmA; for 24 of these patients, anti-tTG assay of the culture medium was positive. The CD patients whose biopsy specimens were positive for serum antibodies showed the following intestinal histologies: total villous atrophy, 35%; severe villous atrophy, 25%; mild atrophy, 25%; villi with no atrophy but with increased intraepithelial lymphocytes, 15%. None

The human mdrl gene encodes a transmembrane glycoprotein the over-expression of which is associated with development of multidrug resistance in human tumor cells. A negative modulation of human mdrt has been attempted via a 27-met unmodified triple helix-forming oligonucleotide, named 1 D, targeted to a homopurine sequence in the coding region of the gene. By administering 10/zM of 1D we could find a significant reduction in MDR1 mRNA levels in the human drug-resistant cell line CEM-VLBI00. This effect appears to be specific and due to a transient block of RNA polymerase mediated by triple helix formation.

We have demonstrated previously that the GT triplex-forming oligodeoxyribonucleotide (TFO) d(TGTGTTTTTGTTTTGTTGGTTTTGTTT), named TFO ID, targeted to a polypyrimidine-polypurine coding sequence located within human multidrug-resistance mdrl gene, specifically and significantly reduced mdrl mRNA levels in the drug-resistant T-leukemic CEM-VLB100 cell line. In this article, we demonstrate that TFO 1D is effective at inhibiting not only transcription but also replication of mdrl genes, leading to a loss of amplified gene copies in the drug-resistant colon adenocarcinoma LoVo DX cell line. In contrast, TFO ID does not alter replication of the constitutive mdrl gene copy in the corresponding parental sensitive LoVo 109 cell line. A specific reduction in mdrl gene amplification levels was also obtained with the pyrimidine TFO d(CTTTTTCTTTTCTTCCTTTTCTTT), named TFO 24TC, directed against the same polypyrimidine-polypurine sequence of the mdrl gene. We suggest that triple helix-forming oligonucleotides might affect the replication of unstable chromosomal elements as amplicons in actively replicating cells by causing a local impairment of DNA polymerase activity. This study lends support to the notion that TFO may be used to reduce gene amplification aiming to control neoplastic progression in cancer cells bearing amplified oncogenes.

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