Effects on tumor cells of ribozymes that cleave the RNA transcripts of human papillomavirus type 18
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1Department of Basic Sciences, University of Texas Dental Branch, Houston 77225, USA.
Abstract:
Many human cervical and oral carcinomas express RNA of human papillomaviruses, and the RNA transcript provides a potential target for gene therapy of these carcinomas. Three hammerhead ribozymes that were targeted to RNA of human papillomavirus type 18 (HPV-18) were cloned into a plasmid expression vector. Each plasmid was then transfected into the HPV-18-expressing cell line. HeLa, or the non-HPV-expressing oral cancer cell line, Tu167. None of the ribozymes had any effect on the phenotype of Tu167 cells. In contrast, each ribozyme affected the phenotype of HeLa cells, causing reduced growth rates, increased serum dependency, and reduced focus formation in soft agar. A molecule that had the same antisense sequences as a ribozyme but lacked the catalytic sequences affected the HeLa cell phenotype to a much lesser extent. The effects of two of the ribozymes could be attributed in part to an increased intracellular concentration of the tumor suppressor protein p53. The most effective ribozyme was targeted to nucleotide 309 in the HPV-18 transcript, but each of the three ribozymes appears to have potential for gene therapy of cancers that express HPV-18.
Insights
Hammerhead ribozymes targeting human papillomavirus type 18 (HPV-18) RNA show potential for cancer gene therapy. These ribozymes effectively reduced growth and altered phenotypes in HPV-18-expressing cancer cells.
Area of Science:
- Molecular Biology
- Virology
- Oncology
Background:
- Human papillomavirus (HPV) RNA is expressed in cervical and oral carcinomas.
- HPV RNA presents a potential therapeutic target for gene therapy.
- Hammerhead ribozymes offer a strategy for targeting specific RNA transcripts.
Purpose of the Study:
- To evaluate the efficacy of hammerhead ribozymes against human papillomavirus type 18 (HPV-18) RNA.
- To assess the potential of these ribozymes for gene therapy in HPV-18-associated cancers.
Main Methods:
- Cloning three hammerhead ribozymes targeting HPV-18 RNA into a plasmid expression vector.
- Transfecting plasmids into HPV-18-expressing (HeLa) and non-expressing (Tu167) cancer cell lines.
- Analyzing phenotypic changes in transfected cells, including growth rate, serum dependency, and soft agar colony formation.
Main Results:
- Ribozymes significantly affected HeLa cell phenotype, reducing growth and focus formation.
- Non-catalytic antisense molecules showed minimal effect on HeLa cells.
- Two ribozymes increased intracellular concentrations of the tumor suppressor protein p53.
- The most effective ribozyme targeted nucleotide 309 of the HPV-18 transcript.
Conclusions:
- Hammerhead ribozymes targeting HPV-18 RNA demonstrate therapeutic potential for HPV-18-expressing cancers.
- Ribozyme efficacy is linked to catalytic activity and specific targeting within the HPV-18 transcript.
- Further development of these ribozymes could lead to novel gene therapies for HPV-associated malignancies.