Detection of p16 gene alteration in cervical cancer using tissue microdissection and LOH study.

The p16 gene was identified as cyclin-dependent kinase inhibitor (CDKI) and this may negatively regulate the cell cycle by acting as a tumor suppressor. Using tissue microdissection, the molecular changes at p16 and Rb genes were analysed in the spectrum of disease from dysplasia to invasive cancer of the uterine cervix. Six of 27 (22%) cases informative for D9S171 and IFNA of 9p21-22 marker (p16INK4a) showed loss of one or both alleles in at least one of these loci. LOH of pRb was detected in 29% (5/17). Gene alterations at p16 and pRb loci were only detectable in some cases of HPV-16/18 DNA positive cervical cancer. Three cases demonstrated mutational changes of p16INK4a, and the alterations were determined to be G to T shift, suggesting transitional missense mutation. In summary, the inactivation of the p16/cdk-cyclin/Rb cascade may play an additional role during the malignant progression in HPV-16/18 positive cervical cancers.

Response of human chorionic gonadotrophin to luteinizing hormone-releasing hormone stimulation in the culture media of normal human placenta, choriocarcinoma cell lines, and in the serum of patients with gestational trophoblastic disease.

In vitro and in vivo responses to luteinizing hormone-releasing hormone (LHRH) stimulation of human chorionic gonadotrophin (hCG) production were evaluated. We cultured placental tissues of ten weeks and term pregnancy, choriocarcinoma tissues, and monolayers of the BeWo cell line, and added serial dilutions of LHRH (1, 5 and 10 micrograms) to the media for five to seven days. In in vivo experiments, 100 micrograms LHRH was intravenously administered to 20 normally cycling women (control group), 27 women who were 'possible remission', and 21 women with 'minimal resistance' to gestational trophoblastic disease. After injection of LHRH, blood samples were collected at 0, 30, 60, 90 and 120 minutes. The concentrations of immunoreactive beta-hCG in in vitro culture media, and in sera from patients, were measured before and after LHRH stimulation by double-antibody radioimmunoassay. These in vitro and in vivo experiments revealed that normal and malignant trophoblastic cells responded to the LHRH stimulation by producing immunoreactive beta-hCG. Therefore, LHRH stimulation may be useful in detecting residual choriocarcinoma cells in gestational trophoblastic disease patients during their periremission periods.

Protein kinase C modulates telomerase activity in human cervical cancer cells.

Telomerase, a ribonucleoprotein reverse transcriptase that extends telomeres of eukaryotic chromosomes is repressed in normal somatic cells but is activated during development and neoplasia. The regulation mechanism of telomerase activity in cancer cells is not clearly known. In this report, a possible affect of PKC on telomerase activity was examined using HeLa and CUMC-6 cervical cancer cell lines. Exposure of cells to PKC inhibitor, bisindolylmaleimide I and Gö6976, and high levels of PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA) resulted in the inhibition of PKC activity in both cells. Telomerase activities were also inhibited by bisindolyl-maleimide I and Gö6976, respectively, in a time-dependent manner. As PKC activity changes in TPA-treated cervical cancer cells, telomerase activities were increased at low dose of TPA and decreased at high dose. The expression levels of human telomerase subunits, human telomerase RNA (hTR) were not influenced by PKC modulating drugs. In contrast, the expression of full-length human telomerase reverse transcriptase (hTERT) was decreased after exposure to bisindolylmaleimide I and Gö6976 in a time-dependent manner. hTERT expression was not affected by low dose of TPA. In contrast, high dose of TPA inhibited hTERT expression level. But the expression patterns of beta-deletion transcript of hTERT after 72 h of treatment with PKC inhibitors or high dose of TPA exposure were not discernable as compared with those of full-length hTERT transcripts to PKC modulating drugs. These results suggest that PKC-modulating drugs altered telomerase activities by affecting full-length hTERT expression profile in human cervical cancers.

A new cell line from human undifferentiated carcinoma of the ovary: establishment and characterization.

A cell line designated CUMO-2 has been established from an undifferentiated ovarian carcinoma. The s.c. injection of cells into nude mice gave rise to fast-growing tumors, while the i.p. route induced a peritoneal carcinomatosis with ascites. Histopathologically, the transplanted s.c. tumors closely resembled the original tumor, but tumors developed in the peritoneal cavity were highly anaplastic. The epithelial nature of the cells was confirmed by ultrastructural analysis. Sequential cytogenetic analyses on early and late passages revealed highly aneuploid tumor cells with consistent structural aberrations of chromosomes 1, 3, 8 and 11. CUMO-2 cells were found to produce CA 125 in vitro and in vivo. Cytosol estrogen receptor (ER) was found but progesterone receptor (PR) was not measured. HLA typing indicated the presence of DR8 and DQw4. A gonadotropin-releasing hormone (Gn-RH) analog inhibited cell growth and Gn-RH receptor mRNA was detected by reverse transcription/polymerase chain reaction in this cell line. Administration of transforming growth factor beta 1 inhibited both cell growth and c-myc mRNA expression. This cell line demonstrated a conformational band shift in exon 7 of the p53 gene. It was a frameshift mutation.

Establishment and characterization of a highly tumorigenic human diploid endometrial cancer cell line.

A new cell line designated CUME-1 has been established from a poorly differentiated endometrial adenocarcinoma of the uterus. This cell line grew well without interruption for more than 88 months and 110 serial passages were successively carried out. The cells were highly tumorigenic in nude mice (85%). Repeated karyotype analyses from early (4th) to late (55th) passages of this cell line revealed a diploid stable clone in each passages without any noticeable structural or numerical aberrations. But from the 80th passage, a subpopulation with reciprocal translocation between chromosomes 1q and 9q consistently appeared and was observed in about 30% of the cells. This cell line is one of the rare examples of experimentally proved tumorigenic cells of human solid tumor origin that retains the diploid karyotype in vitro. HLA typing indicated the presence of DR4, DR13, DQ3 and DQ6. Cytosol estrogen and progesterone receptors were found both in fresh primary tumor and in this cell line. Gonadotropin-releasing hormone (Gn-RH) receptor mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) in cultured cells. Using the single-strand conformation polymorphism (SSCP) technique, we have screened CUME-1 cells for p53 mutation in exons 4 to 9. No mobility shift was observed. This cell line may be useful in studying the in vitro chromosomal evolution of the cell line and the in vivo properties of human endometrial adenocarcinoma.

Protective effects of green tea extracts (polyphenon E and EGCG) on human cervical lesions.

We investigated clinical efficacy of green tea extracts (polyphenon E; poly E and (-)-epigallocatechin-3-gallate [EGCG]) delivered in a form of ointment or capsule in patients with human papilloma virus (HPV) infected cervical lesions. Fifty-one patients with cervical lesions (chronic cervicitis, mild dysplasia, moderate dysplasia and severe dysplasia) were divided into four groups, as compared with 39 untreated patients as a control. Poly E ointment was applied locally to 27 patients twice a week. For oral delivery, a 200 mg of poly E or EGCG capsule was taken orally every day for eight to 12 weeks. In the study, 20 out of 27 patients (74%) under poly E ointment therapy showed a response. Six out of eight patients under poly E ointment plus poly E capsule therapy (75%) showed a response, and three out of six patients (50%) under poly E capsule therapy showed a response. Six out of 10 patients (60%) under EGCG capsule therapy showed a response. Overall, a 69% response rate (35/51) was noted for treatment with green tea extracts, as compared with a 10% response rate (4/39) in untreated controls (P<0.05). Thus, the data collected here demonstrated that green tea extracts in a form of ointment and capsule are effective for treating cervical lesions, suggesting that green tea extracts can be a potential therapy regimen for patients with HPV infected cervical lesions.

Regulation of cell growth and HPV genes by exogenous estrogen in cervical cancer cells.

Human papillomavirus (HPV) infection is known as the major cause of the development of cervical cancer. The E6 and E7 proteins of oncogenic HPV can play critical roles in immortalization and malignant transformation of cervical epithelial cells. From the previous epidemiologic data, it has been determined that long-term use of oral contraceptives may be a risk factor for cervical cancer. Investigation of the estrogenic and antiestrogenic effects on the proliferation of cervical cancer cells and the gene expression of HPV would help to explain the role of estrogen in the HPV-associated pathogenesis of cervical cancer. In this study, cervical cancer cells (HeLa, CaSki, and C33A) were cultured in vitro in the presence of 17beta-estradiol or tamoxifen to observe their regulatory growth effect and HPV E6/E7 gene expression. The estrogenic effect on the promoter activity of HPV URR was further confirmed by transient transfection assay, which was conducted in C33A cells using the HPV-18 URR-CAT reporter plasmid. The supplemental effect of estrogen receptors on URR promoter activity was also evaluated. The proliferation of HeLa and CaSki cells was stimulated by estradiol at physiologic concentration levels (

Detection of c-K-ras point mutation in ovarian cancer.

Point mutations of c-K-ras in ovarian cancer were detected by replacement of GGT of codon 12 by GAT, AGT, TGT and GTT, polymerase chain reaction, agarose gel electrophoresis and Southern blot hybridization with a digoxigenin detection system. The incidence of four-typed point mutations of c-K-ras oncogene in 37 ovarian cancers was 35.1% (13/37) and the distributions were 32.4% (12/37), 2.7% (1/37), 0% and 0% of GGT to GAT, GGT to AGT, GGT to TGT, and GGT to GTT, respectively. The incidence of c-K-ras point mutations on codon 12 among 37 patients with ovarian cancer was 35.5% (8/22) in those with serous cystadenocarcinomas and 28.6% (2/7) in those with mucinous cystadenocarcinomas. c-K-ras point mutations on codon 12 were detected in 14.3% (1/7) of patients with stage I disease, 28.6% (2/7) with stage II disease, and in 43.5% (10/23) with stage III/IV disease, and there was a statistically significant increase in point mutations of c-K-ras oncogene with advancing clinical stage. The incidence of c-K-ras point mutations on codon 12 among 33 patients who had a pelvic lymph node dissection was 52.4% (11/21) in those with pelvic lymph node metastases and 16.7% (2/12) in those without pelvic lymph node metastases, a statistically significant difference. Furthermore, point mutation of c-K-ras gene was found most frequently in patients with advanced stage disease, and in those with pelvic lymph node metastases. Activation of c-K-ras oncogene seems to be a major factor in ovarian carcinogenesis and tumor progression.

The antibody response to HPV proteins and the genomic state of HPVs in patients with cervical cancer.

Human papillomavirus (HPV) DNAs are often found to be integrated into the human genome in high-grade cervical intraepithelial neoplasia (CIN) as well as in invasive cervical cancers. Investigation of the relationship between the genomic status of specific HPV genes and their antibody responses to the virus-like particles (VLPs) of HPV-16 L1/L2 proteins and the in vitro translated HPV-16 E6 and E7 proteins may help to illustrate the mechanism of HPV-related cervical carcinogenesis and host immune response. Cervical cancer tissues obtained from 39 patients were studied to evaluate the physical status of HPV genes by Southern blotting, DNA-PCR, and RT-PCR of E2. The antibody response against the HPV-16 L1/L2 VLPs of serum specimens were tested by ELISA and the antibody response against the HPV-16 E6 and E7 proteins were tested by radioimmunoprecipitation assay (RIPA), respectively. Integrated forms of HPV-16 DNA were found in 23 of the 38 patients (60.5%). The HPV-16 positive cervical cancer patients showed a significantly higher prevalence rate (39.5%; 15/38) of antibodies to HPV-16 L1/L2 VLPs than that of the control group (8.7%; 2/28) (P < 0.05). Antibodies to HPV-16 L1/L2 VLPs were more commonly detectable in cervical cancer patients having the episomal form of HPV-16 DNA (pure episomal and mixed forms) (60%; 9/15) than in those who had only the integrated forms of HPV-16 DNA (26.1%; 6/23) (P < 0.05). Antibodies to E6 and E7 proteins were positive in 36.8% (14/38) and 50% (19/38) of the patients with HPV-16 positive cervical cancer, respectively. These were significantly higher than the positive rates for the control group (8.3% and 2.8%) (P < 0.05). The differences between sero-reactivities to E6 and E7 proteins in the patients with episomal forms of HPV-16 DNA and those with integrated forms of HPV-16 DNA were not statistically significant (P > 0.05). Integrated forms of HPV-16 DNA were prevalent in most patients with cervical cancer in Korea. Antibodies to HPV-16 L1/L2 VLPs, in vitro translated HPV-16 E6 and E7 proteins, appeared in a significantly larger proportion of the HPV-associated cervical cancer patients than in the controls. Antibodies to HPV-16 L1/L2 VLPs were more often detected in cervical cancer patients having the episomal form of HPV-16 DNA than in those having only integrated forms of HPV-16 DNA. Antibody responses to HPV-16 E6 and E7 proteins were not influenced by the different viral states.

Clinical application of HPV typing in cervical cancer.

Certain human papaillomavirus (HPV) types are major risk factors for the development of cervical neoplasia. The value of HPV DNA testing in the management of patients with disease and in population screening is a subject of controversy. Since the introduction of molecular biology into the HPV field, there have been rapid advances and improvements in HPV diagnosis. The various molecular diagnostic methods for detection of HPV DNA (dot blot hybridization, Southern blot hybridization, in situ hybridization, Hybrid Capture Test, and polymerase chain reaction; PCR) could be selected by taking into consideration some factors such as characteristics of sample, sensitivity of HPV test and expenses. The HPV DNA testing would be a clinically useful diagnostic method, when used in conjunction with the Pap smear in population screening or in conjunction with cytology and colposcopy to identify the women infected with high-risk HPVs or women who had equivocal cervical lesions. Despite the confusion, a multitude of reports demonstrate that HPV DNA testing has the clinical utility, and future investigations should be directed at more accurately delineating its role in human health care.

In vivo release of beta human chorionic gonadotropin by luteinizing hormone releasing hormone stimulation and its clinical application as a remission criterion in patients with gestational trophoblastic disease.

In vivo responses of trophoblasts to luteinizing hormone releasing hormone (LHRH) stimulation in 48 gestational trophoblastic disease patients were observed. Serum beta human chorionic gonadotropin (beta-hCG) levels after LHRH injection were significantly increased in patients with hCG values between 5 and 20 mIU/ml (minimal resistance group) but not in patients whose hCG levels were less than 5 mIU/ml (possible remission group). The sensitivity, specificity and the predictive values of LHRH stimulation test were 75.0, 91.3 and 95.5% in the possible remission group and 87.5, 20.0 and 77.8% in the minimal resistance group.

Persistence of human papillomavirus as a predictor for treatment failure after loop electrosurgical excision procedure.

We aimed to investigate whether postconization human papillomavirus (HPV) DNA testing can predict treatment failure and improve the accuracy of conventional follow-up in women with high-grade cervical intraepithelial neoplasia (CIN). Between March 2001 and October 2005, 120 patients with confirmed CIN 2 or 3 were treated with loop electrosurgical excision procedure (LEEP) and were enrolled. Six patients were lost to the follow-up. Postconization follow-up was performed at every 3-6 months during the first year and then annually. Specimens were tested for the presence of HPV, using the Hybrid Capture 2 (Digene Co, Gaithersburg, MD) and HPV DNA chip (Mygene Co, Seoul, Korea) test. Persistent HPV infection was defined as persistently (two times or more) positive HPV tests with the same HPV subtype(s) at initial diagnosis. Twenty-two (19.3%) patients showed treatment failure after conization. The only significant risk factor for redevelopment of CIN after conization was persistence of the same HPV subtype (P < 0.0001). And women with recurrent or residual CIN had higher HPV load during the 6-month follow-up postconization. In conclusion, the persistence of the same HPV subtype after LEEP conization was an important predictor of treatment failure. The follow-up protocol after conization of CIN should include both cervical cytology and HPV test, and HPV DNA chip test is needed to detect a persistent HPV infection.

Human papillomavirus genotype as a prognostic factor in carcinoma of the uterine cervix.

The clinical implications of specific human papillomavirus (HPV) types in invasive cervical carcinomas are only now beginning to be appreciated. The objective of this study was to determine the clinical implications and prognostic value of the HPV genotype in cervical carcinomas. In this study, we employed an HPV DNA chip to detect the type-specific sequence of HPV from cervical swabs taken from women with biopsy-proven neoplastic lesions of the cervix. We divided the patients into four groups: HPV-negative, HPV-16-related, HPV-18-related, and intermediate risk type-related. Associations with clinicopathologic data (stage, histologic type, lymph node status, parametrial invasion, lymphvascular space invasion, tumor size, vaginal involvement) and overall survival were assessed. HPV DNA was detected in 81.4% of the patients, and 19.0% harbored multiple HPV variants. HPV-16-related was the predominant type and was detected in 47.4% (46/97) of the patients. The HPV-16-related types were detected more frequently in patients with squamous cell carcinomas, whereas the HPV-18-related types were more prevalent in cases of adenocarcinomas and adenosquamous carcinomas (P < 0.05). Otherwise, no significant correlations were detected between the HPV genotype and any other clinicopathologic parameters. After a median follow-up of 30 months, the 5-year survival rate was lower in the HPV-18-related patients, but this difference was not found to be statistically significant, according to the results of the log-rank test. We conclude that neither the presence nor type of HPV DNA bears any prognostic significance in cases of cervical carcinoma.

Development of anticancer gene vaccine interact with human papillomavirus oncoprotein inhibition.

Adeno-associated virus (AAV) Rep 78 protein is known to inhibit the promoter site of several oncogenes and viral genes, including the human papillomavirus (HPV) type 16 E6 transforming genes. The biochemical studies of Rep 78 have been reported, but the effects of Rep 78 gene-mediated inhibition of HPV 16 E6 promoter activity on the various human cervical carcinoma cells have not been characterized. pEGFP-N1 vector, cloned by AAV-mediated Rep 78, is transfected into cervical carcinoma cells. Transfection efficiency of Rep 78 was approximately 30-60% different. Messenger RNA (mRNA) and protein expression of Rep 78 gene was significantly higher on day 1 of the transfection of Rep 78 DNA in CaSki cells, and DNA level of HPV 16 E6 was decreased on day 1 of the transfection. The growth of CaSki cervical cancer cells was only 10-15% inhibited by Rep 78, and the other cervical cells, HeLa, HeLaS3, HT3, and QGU, were unaffected by Rep 78 transfection. In spite of the high efficiency of Rep 78 gene transformation and expression rate, we could not show the significant growth inhibition in various cervical cancer cell lines. Taken together, long-term expression of Rep 78 strategy might be needed for cervical carcinoma gene therapy using AAV vector.

Distinctive cell cycle regulatory protein profiles by adenovirus delivery of p53 in human papillomavirus-associated cancer cells.

In this study, microarray analyses were performed to determine the time course of gene expression profiles in SiHa cells after infection with an adenovirus-expressing p53 (Adp53). We then investigated the consequences of Adp53 gene transfer on the expression level of six genes associated with cell cycle control and on apoptosis and cell cycle arrest in SiHa cells and compared these results with those from CaSki and HeLa cells. Gene expression profiling of the p53-targeted genes in SiHa cells revealed that p21, p53, and mdm2 protein expression was significantly upregulated at 24 and 48 h. Western blot results revealed that p21 and p53 expression levels had significantly increased after Adp53 infection. Cyclin-dependent kinase 4 levels were decreased 48 h after treatment in SiHa and CaSki cells. Proliferating cell nuclear antigen levels were unchanged after Adp53 infection. Only SiHa cells exhibited significant cell death. Cell cycle arrest at the G1 phase was induced in the SiHa and HeLa cells but was not induced at the G2/M and S phases in the CaSki cells. These data support the notion that the understanding of p53-dependent apoptosis and cell growth arrest could be applicable to advanced strategies in the development of preferential tumor cell-specific delivery.

Cellular process classification of human papillomavirus-16-positive SiHa cervical carcinoma cell using Gene Ontology.

This study utilized mRNA differential display and the Gene Ontology (GO) analysis to characterize the multiple interactions of a number of genes involved in human papillomavirus (HPV)-16-induced cervical carcinogenesis. We used HPV-16-positive cervical cancer cell line (SiHa) and normal human keratinocyte cell line (HaCaT) as a control. Each gene has several biological functions in the GO, and hence, we chosen the several functions for each gene. and then, the specific functions were correlated with gene expression patterns. The results showed that 157 genes were up- or down-regulated above two-fold and organized into mutually dependent subfunction sets depending on the cervical cancer pathway, suggesting the potentially significant genes of unknown function. The GO analysis suggested that cervical cancer cells underwent repression of cancer-specific cell-adhesive properties. Also, genes belonging to DNA metabolism such as DNA repair and replication were strongly down-regulated, whereas significant increases were shown in protein degradation and in protein synthesis. The GO analysis can overcome the complexity of the gene expression profile of the HPV-16-associated pathway and identify several cancer-specific cellular processes as well as genes of unknown function. Also, it can become a major competing platform for the genome-wide characterization of carcinogenesis.

Antiproliferative effects of retinoic acid/interferon in cervical carcinoma cell lines: cooperative growth suppression of IRF-1 and p53.

Retinoids and interferons have been implicated in the growth regulation of cervical cancer cells. However, the molecular mechanisms are not fully defined. To analyze detailed mechanisms, HPV-positive (HeLa, CaSki), HPV-negative (C33A, HT-3) and non-cervical Cos-1 cell lines were treated with I microM all-trans-retinoic acid (RA) and/or 10 ng/ml interferon-gamma (IFN-gamma). The growth of RA-treated HeLa cells was less effectively suppressed than that of IFN-gamma-treated ones. A combination of RA and IFN-gamma leads to an additive antiproliferative effect on the cell growth. CaSki cell growth was also inhibited by IFN-gamma but was little stimulated by RA treatment, and the IFN effect was attenuated when IFN-gamma was combined with RA. HPV-negative C33A and HT-3 cells, which are defective in p53 and Rb, and Cos- 1 cells were weakly or not responsive to all combined treatments. The molecular mechanism underlying the differential effects of RA/IFN on HeLa and C33A cells was investigated. Combined RA/IFN-gamma treatment caused a marked increase in the level of IFN regulatory factor-1 (IRF-1) in HeLa, whereas no induction of IRF-1 was observed in C33A, consistent with the findings that IFN signaling is functional in HeLa but is defective in C33A cells. The increase of p53 in HeLa cells might account for the down-regulation of HPV-18 E6 gene expression by RA/IFN-gamma. Furthermore, RA/IFN-gamma treatment resulted in the concurrent induction of p21WAF1 CDK inhibitor and dephosphorylation of Rb protein. Transient co-expression of IRF-1 and p53 led to the cooperative activation of the p21WAF1 promoter. Our results indicate that 2 transcription factors, increased in response to RA/IFN-gamma, cooperate functionally to regulate the cell cycle through the activation of a common p21WAF1 gene in HeLa cells.

Inactivation of interferon regulatory factor-1 tumor suppressor protein by HPV E7 oncoprotein. Implication for the E7-mediated immune evasion mechanism in cervical carcinogenesis.

In studying biological roles of interferon regulatory factor (IRF)-1 tumor suppressor in cervical carcinogenesis, we found that HPV E7 is functionally associated with IRF-1. Binding assays indicate a physical interaction between IRF-1 and HPV E7 in vivo and in vitro. The carboxyl-terminal transactivation domain of IRF-1 was required for the interaction. Transient co-expression of E7 significantly inhibits the IRF-1-mediated activation of IFN-beta promoter in NIH-3T3 cells. Co-transfection of E7 mutants reveals that the pRb-binding portion of E7 is necessary for the E7-mediated inactivation of IRF-1. It was next determined whether histone deacetylase (HDAC) is involved in the inactivation mechanism as recently suggested, where the carboxyl-terminal zinc finger domain of E7 associates with NURD complex containing HDAC. When trichostatin A, an inhibitor of HDAC, was treated, the repressing activity of E7 was released in a dose-dependent manner. Furthermore, the mutation of zinc finger abrogates such activity without effect on the interaction with IRF-1. These results suggest that HPV E7 interferes with the transactivation function of IRF-1 by recruiting HDAC to the promoter. The immune-promoting role of IRF-1 evokes the idea that our novel finding might be important for the elucidation of the E7-mediated immune evading mechanism that is frequently found in cervical cancer.

Comparison of L1 consensus primers with E6 type specific primers for detection of human papillomaviruses in paraffin sections of cervical neoplasia.

In our comparative study of L1 consensus primers with E6 type-specific primers for detection of human papillomavirus (HPVs) by polymerase chain reaction (PCR) in 35 cases of cervical neoplasia, the detection rate by E6 primers (54%; 19/35) was significantly higher than that by L1 primers (25%; 9/35) (p < 0.01). And all specimens HPV-positive with L1 primers were also positive by E6 primers. HPV DNA could be amplified in 36% (9 of 25) of tissue by L1 consensus primers from which beta-globin gene was amplified as compared with 64% (16 of 25) of tissue by E6 type-specific primers. With the L1 consensus primers, 8 cases were positive for HPV-16 and 1 case was positive for HPV-33. These results show that the L1 consensus primers have inferior sensitivity to the E6 type-specific primers for the detection of HPV by PCR. But the L1 consensus primers have great value in making simultaneous detection of various HPV types in a single tube reaction, thus they permit reduction of time and the economic burden of the experiment.

Detection of human papillomavirus genotypes in cervical neoplasia from Korean women using polymerase chain reaction.

The histopathologic features of cervical dysplasia (20 cases) and cervical carcinoma (15 cases) in Korean women were correlated with the presence of human papillomaviruses (HPVs) as determined by polymerase chain reaction technology on paraffin-processed specimens. A segment of the E6 open reading frame of several HPV types (HPV 6, 11, 16, 18, 31, 33, and 35) was amplified using primers which were synthesized to contain 50% G + C, in order to give optimum annealing for the amplification of HPVs. All specimens were also tested for amplification of the cellular beta-globin gene. HPV was found in 19 (54%) cases. The HPV types were HPV 6 in 1 case (3%), HPV 16 in 15 cases (43%), HPV 18 in 2 cases (6%), and HPV 33 in 3 cases (9%). HPV was identified in 16 of 25 (64%) beta-globin-positive and 3 of 10 (30%) beta-globin-negative tissues. HPV types 11, 31, and 35 were not detected in any of the specimens. HPV 6, 18, and 33 were detected only in preneoplastic lesions, and co-infection with these three viruses was observed in one case of severe dysplasia. HPV type 16 was found in 8 (40%) premalignant lesions and in 7 (47%) invasive carcinomas. These data indicate that HPVs are found along the entire spectrum of cervical neoplasia. HPV 16 was the predominant virus in invasive cancer of the cervix.