Analysis by polymerase chain reaction of the physical state of human papillomavirus type 16 DNA in cervical preneoplastic and neoplastic lesions.
Integration of human papillomavirus (HPV) DNA into the host cell genome is believed to be essential for malignant progression. However unambiguous detection of the physical state of HPV is a difficult and time-consuming procedure. To resolve this issue a simple, rapid and highly sensitive technique of polymerase chain reaction (PCR) has been utilized for detecting the physical state of HPV-16 DNA. Investigations were carried out in 122 cervical specimens comprising the whole spectrum of cervical lesions starting from cervical dysplasia to invasive carcinoma including HPV-16-positive normal controls. A pair of oligonucleotide primers specific to the E2 open reading frame, which is often deleted or disrupted following HPV integration, was used for the study. Distinction between episomal and integrated forms of viral DNA was accomplished by detecting amplification of the E2-specific fragment (1139 bp) in the PCR product. The PCR results were compared with those obtained by the conventional methods of Southern blotting, two-dimensional gel electrophoresis and chromosomal in situ hybridization; a high degree of agreement was observed between the methods. The findings indicate that although integrated forms of HPV-16 DNA were detected in more than 70% of cervical cancer specimens, integration was less frequent (23%) in severe dysplasia and carcinoma in situ. Only 2.5% of cases showed both episomal and integrated forms of HPV-16 DNA. The difference between episomal and integrated forms was statistically significant (P less than 0.01). The absence of integration in about 30% of cancer cases suggests that integration of HPV may not be necessary for malignant progression and alternative mechanism(s) of malignant transformation may occur without HPV integration. The PCR test thus provides an effective complement to Southern blotting and two-dimensional gel electrophoresis for accurate detection of the integration of HPV DNA.
Das
B C
BC
Division of Molecular Oncology, Maulana Azad Medical College Campus, New Delhi, India.
Sharma
J K
JK
Gopalakrishna
V
V
Luthra
U K
UK
D001483:Base Sequence; D002277:Carcinoma / Q000382:microbiology*; D002471:Cell Transformation, Neoplastic; D002584:Cervix Uteri / Q000473:pathology; D002877:Chromosomes, Human; D004279:DNA, Viral / Q000302:isolation & purification*; D004268:DNA-Binding Proteins / Q000235:genetics; D005260:Female; D005784:Gene Amplification; D006801:Humans; D008969:Molecular Sequence Data; D027383:Papillomaviridae / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011230:Precancerous Conditions / Q000382:microbiology*; D014412:Tumor Virus Infections / Q000235:genetics*; D002583:Uterine Cervical Neoplasms / Q000382:microbiology*; D014764:Viral Proteins / Q000235:genetics; D016662:Virus Integration
D004279:DNA, Viral; D004268:DNA-Binding Proteins; C051550:E2 protein, Bovine papillomavirus; D014764:Viral Proteins