The inhibition of tumor growth by triplex-forming oligonucleotides
Introduction
Disordered p53 function is commonly encountered in human tumors which consequently escape apoptosis [1]. Novel methods for restoring p53 function in p53-deficient tumor cells have recently been reported [2]. Some years ago a p53 DNA-binding consensus sequence was identified that is present in the human nontranscribed ribosomal spacer region [3]. Given the potential participation of ribosomal gene transcription in cellular proliferation we elected to assess the effect of functionally substituting for p53 binding to this site [4], [5]. We developed several oligonucleotides in an effort to achieve this goal, one of which was designed to produce triple helical structures by Hoogsteen hydrogen bond base pairing at the target sequence [5], [6]. Gel shift studies confirmed binding of oligonucleotide Hoog 1 to the target sequence and cell culture studies revealed that application of this oligonucleotide markedly reduced the proliferation of COLO 201 and HCT 116 colon carcinoma cells—cells which contain mutant p53 protein. Cell death did not occur. The proliferation of normal human lung fibroblasts was reduced much less [5]. We now extend these observations to the suppression of tumor growth in a nude mouse/subcutaneous tumor model. HCT 116 cells were used because they readily formed tumors in the mice. Although these cells contain normal p53 protein, our earlier studies revealed they also contain mutant p53 and we previously showed that Hoog 1 reduced the growth of these cells in vitro [5], [7].
Section snippets
Materials and methods
Pathogen-free four-to-six week old female Ncr-nude mice were obtained from Taconic (Germantown NY) and were housed in sterilized, filter-topped cages kept in a laminar flow isolator (Forma Scientific, Marietta, OH). Mice were fed autoclaved food and water ad libitum. Thirty mice were injected subcutaneously with 2×106 HCT 116 human colon carcinoma cells into the dorsal flanks. Tumors were palpable generally within 1–2 weeks. Palpable tumors were injected with test reagent within one day of
Results
Final (14 day) tumor size means and standard deviations for the treatment groups are shown in Table 1, and tumor growth curves are shown in the Fig. 1 (note that the ordinate scales differ in the three panels). The daily administration of triplex-forming oligonucleotide Hoog 1 produced a statistically significant reduction in tumor growth when compared either to vehicle or inactive oligonucleotide (Hoog 3). ANOVA Testing of log transformed data revealed that the three groups are significantly
Conclusions
These results demonstrate the capacity of triplex-forming oligonucleotides directed at a consensus p53 binding sequence to reduce the growth of colon cancer cells in vivo. This was not a non-specific effect because a control oligonucleotide (Hoog 3) was without effect, just as it was without effect in our earlier cell culture studies [5]. Moreover, tumor suppression by the active oligonucleotide was marked, effectively eliminating tumor growth above baseline (data not shown). Tumor shrinkage
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