In the United States, cervical cancer affects approximately 8.6 women per 100,000 each year. Human Papillomaviruses (HPVs) are frequently associated with latent infections, benign and premalignant cervical lesions, and half of the invasive cervical carcinomas. The oncogenic potential of HPVs appears to be associated with the products of two viral genes. Our first goal is to interrupt E6/E7 which is required for the acquisition and maintenance of a transformed phenotype. If E6/E7 are eliminated, carcinoma cells should die. The second goal is to develop a hairpin ribozyme technique for gene disruption. For this purpose, a hairpin ribozyme (originally from the negative strand of the tobacco ring spot virus) was developed to cleave the E6 gene. Hairpin ribozymes are more efficient than hammerhead ribozymes at 37 degrees. The target must be a GUC site; six exist in the E6 gene. The fidelity of these sites was proven by examining 78 HPV-16 cervical carcinomas from Caucasian and Hispanic patients by polymerase chain reaction linked to single stranded conformational polymorphism (SSCP). Analysis indicated that nine DNA samples had heterozygous mutations within the same region of the E6 gene 3' terminus; T C transitions at HPV-16 position 511 (silent) and one of the nine also had a 513 mutation (Met Thr). These mutations do not affect the E6 structure or function, although a correlation was found between the presence of a mutation at base 511 and the metastatic potential of the carcinoma and poor survival of the patients. The ribozyme was found effective in cleaving 95 percent of a synthetic E-6. Therefore, it was placed in a retrovirus and tested for effectiveness using CXT-1 cell line derived from a spontaneous HPV-16 cervical carcinoma. Proof that the ribozymes are found in cervical cells was demonstrated using the RNase protection technique and a probe complementary to the ribozyme. The ribozyme in the antisense configuration was not found in cells. The ability of the ribozyme to cleave E6 mRNAs was shown in ten-day old cultures by reverse transcription polymerase chain reaction. Research is underway to increase the efficiency of destruction of the E6 gene. Additional studies involve the use of antisense molecules to inhibit the growth of HPV-16 positive cells.
