Role In Cervical Carcinogenesis Research Articles

An association between human papillomavirus 16/18 deoxyribonucleic acid in peripheral blood with p16 protein expression in neoplastic cervical lesions

Background: Human papillomavirus (HPV) infection plays a crucial role in cervical carcinogenesis. Apart from the detection of p16 protein in cervical tissues, the feasibility of the presence of HPV DNA in peripheral blood being an auxiliary marker of cervical lesions was examined. Methods: Peripheral blood samples and cervical tissues, from 36 cervical tissues from high-grade squamous intraepithelial lesions (HSIL) and 31 early invasive cervical cancers (EICC), were analyzed for HPV 16/18 DNA and HPV 16/18 E7 mRNA expression, as well as the in situ expressions of p16 and pRb to investigate the in-between associations. Results: The prevalence of HPV 16/18 DNA in patients with EICC was relatively higher than those of HSIL, in both of cervical tissues and peripheral blood. The presence of HPV 16/18 DNA in peripheral blood was positively correlated with that in cervical tissue, as well as with p16 overexpression in cervical tissues together with a significant correlation between E7 mRNA and pRb and p16 protein expressions. Discussion: A positive correlation between the presence of HPV 16 or 18 DNA in peripheral blood and p16 overexpression in tissues of patients with cervical lesions was confirmed. Together with p16 immunostaining in cervical tissues, the detection of high-risk HPV 16 or 18 DNA in peripheral blood may act as an auxiliary biomarker for HPV-associated neoplastic cervical lesions.

Epstein-Barr Virus and p16INK4A Methylation in Squamous Cell Carcinoma and Precancerous Lesions of the Cervix Uteri

Methylation of p16 is an important mechanism in cervical carcinogenesis. However, the relationship between cervical squamous cell carcinoma (SCC) and Epstein-Barr virus (EBV) remains controversial. Here, we explored whether EBV infection and/or p16 gene inactivation would play any role in cervical carcinogenesis. Eighty-two specimens included 41 invasive SCCs, 30 cervical intraepithelial neoplasm (CIN; CIN 1, 11 cases, CIN II, 3 cases, CIN III 16 cases) and 11 nonneoplastic cervices. EBV was detected by polymerase chain reaction (PCR) for EBNA-1 and in situ hybridization for EBER-1. The p16 methylation-status and the expression of p16 protein were studied by methylation-specific PCR and immunohistochemistry, respectively. The materials were divided into four groups: 1) nonneoplastic cervices, 2) CIN I, 3) CIN II-III and 4) invasive SCCs. p16 methylation and p16 immunoexpressions increased in CIN and invasive SCCs than nonneoplastic tissue. p16-methylation and p16-immunoreactivities were higher in the EBV-positive group (p=0.009, p<0.001) than in the EBV-negative group. EBV was detected more frequently in CIN and SCCs than nonneoplastic cervices. In conclusion, a correlation between p16 methylation, p16 immunoreactivity and the detection of EBV strongly suggested that the cooperation of EBV and p16 gene may play a synergic effect on cell cycle deregulation.

Nested PCR with the PGMY09/11 and GP5 +/6 + primer sets improves detection of HPV DNA in cervical samples

Based on epidemiological and research evidence, HPV has a causal role in cervical carcinogenesis. Several HPV detection methods exist to date; the most commonly used method for detection of genital HPVs consists of nested PCR using the MY09/11 and GP5 +/6 + primer sets (MY/GP +). Recently, the PGMY09/11 primer set, a modified version of the MY09/11 primer set, was introduced for single PCR and was found to detect a wider range of HPV types. The next logical step was taken and the efficacy of nested PCR using the PGMY09/11 and GP5 +/6 + primer sets (PGMY/GP +) to detect HPV in cervical samples was evaluated. In this comparative study, nested PCR using the novel PGMY/GP + primer set combination was found to be more type sensitive than the nested PCR with the MY/GP + primer sets, detecting a wider range of HPV types, low copy HPVs, and better characterizing samples infected with multiple strains of HPV. Standardization and use of the PGMY/GP + PCR system could aid physicians in providing more efficient HPV screening and better treatment for patients.

High level HPV-16 E7 oncoprotein expression correlates with reduced pRb-levels in cervical biopsies.

High-risk human papillomaviruses (HPVs) are major etiological agents of cervical cancer. Despite excellent epidemiological evidence for a direct role of HPV-16 in cervical carcinogenesis, molecular pathways underlying carcinogenesis in vivo remain obscure. The E7 gene is required for immortalization and maintenance of the transformed phenotype in vitro; however, little is known about its role for tumorigenesis in vivo. The E7 gene codes for an unstable protein the abundance of which in cervical biopsies is unknown. We show here that E7 protein levels strongly increase during cervical carcinogenesis, underlining its fundamental role in cervical cancer. The E7 protein was found predominantly in the nucleus and to a minor extent in the cytoplasm in the cervical cancer cell line Ca Ski in vitro and in invasive cervical carcinoma in situ, suggesting that nuclear resident E7 plays a major role in cervical carcinogenesis in humans. The retinoblastoma protein (pRb) is a major E7-target in vitro. We show here that pRb expression is initially upregulated in LSIL and disappears in later stages concomitant with increased E7 levels, suggesting that E7-driven degradation of pRb is involved in cervical tumorigenesis in humans.

Expression of a novel human gene, human wings apart-like (hWAPL), is associated with cervical carcinogenesis and tumor progression.

In Drosophila melanogaster, the wings apart-like (wapl) gene encodes a protein that regulates heterochromatin structure. Here, we characterize a novel human homologue of wapl (termed human WAPL; hWAPL). The hWAPL mRNA was predominantly expressed in uterine cervical cancer, with weak expression in all other normal and tumor tissues examined. hWAPL expression in benign epithelia was confined to the basal cell layers, whereas in dysplasias it increasingly appeared in more superficial cell layers and showed a significant correlation with severity of dysplasia. Diffuse hWAPL expression was found in all invasive squamous cell carcinomas examined. In addition, NIH3T3 cells overexpressing hWAPL developed into tumors on injection into nude mice. Furthermore, repression of hWAPL expression by RNA interference induced cell death in SiHa cells. These results demonstrate that hWAPL is associated with cell growth, and the hWAPL expression may play a significant role in cervical carcinogenesis and tumor progression.

Establishment of a HPV and p53-mutation-negative human cell line (CA) derived from a squamous carcinoma of the uterine cervix

Objective. Well-characterized human cancer cell lines are important research resources for studying cancer cell biology, as well as for developing new strategies against cancer cell growth and progression. We present a new cell line, CA, established from an invasive non-keratinizing squamous cell carcinoma of the uterine cervix in 36-year-old patient. Methods. We measured the doubling time of CA cells. To investigate the tumorigenicity of CA, cells were inoculated subcutaneously into the back of nude mice. Several tumor markers were analyzed using culture media by EIA. PCR-based analyses were performed to examine the human papillomavirus (HPV) status and telomerase activity. CA was also screened for p53 mutation using the sequencing technique. Results. The cells show rapid growth in culture with a doubling time of 14.3 h and high migration activity. Monolayer-cultured cells were polygonal, showing a pavement-like arrangement and a tendency to pile up without contact inhibition. Subcutaneous transplantation of the CA cells into nude mice formed solid tumors that were histologically diagnosed as squamous cell carcinoma, whereas no metastasis was observed. Cultured CA cells produced SCC, CEA, TPA, CA125 and SLX. Genetic and molecular analyses revealed high telomerase activity and the absence of HPV DNA. No p53 mutation was observed in this cell line. Conclusion. These properties suggest that CA is an aggressive cervical carcinoma cell line and may serve as a useful experimental model for studying HPV role in cervical carcinogenesis.

KMP025 Application of a quantitative fragment analysis technique (T-RFLP) technique in medical microbiology

Based on epidemiological and research evidence, HPV has a causal role in cervical carcinogenesis. Several HPV detection methods exist to date; the most commonly used method for detection of genital HPVs consists of nested PCR using the MY09/11 and GP5+/6+ primer sets (MY/GP+). Recently, the PGMY09/11 primer set, a modified version of the MY09/11 primer set, was introduced for single PCR and was found to detect a wider range of HPV types. The next logical step was taken and the efficacy of nested PCR using the PGMY09/11 and GP5+/6+ primer sets (PGMY/GP+) to detect HPV in cervical samples was evaluated.In this comparative study, nested PCR using the novel PGMY/GP+ primer set combination was found to be more type sensitive than the nested PCR with the MY/GP+ primer sets, detecting a wider range of HPV types, low copy HPVs, and better characterizing samples infected with multiple strains of HPV. Standardization and use of the PGMY/GP+ PCR system could aid physicians in providing more efficient HPV screening and better treatment for patients.

Hypoxia-Inducible Erythropoietin Signaling in Squamous Dysplasia and Squamous Cell Carcinoma of the Uterine Cervix and Its Potential Role in Cervical Carcinogenesis and Tumor Progression

Tissue hypoxia is a characteristic property of cervical cancers that makes tumors resistant to chemo- and radiation therapy. Erythropoietin (Epo) is a hypoxia-inducible stimulator of erythropoiesis. Acting via its receptor (EpoR), Epo up-regulates bcl-2 and inhibits apoptosis of erythroid cells and rescues neurons from hypoxic damage. In addition to human papillomavirus infection, increased bcl-2 expression and decreased apoptosis are thought to play a role in the progression of cervical neoplasia. Using reverse transcriptase-polymerase chain reaction and Western blotting we showed that HeLa and SiHa cervical carcinoma cells and human cervical carcinomas express EpoR, and that hypoxia enhances EpoR expression. Exogenous Epo stimulated tyrosine phosphorylation and inhibited the cytotoxic effect of cisplatin in HeLa cervical carcinoma cells. Using immunohistochemistry, we examined the expression of Epo, EpoR, p16, hypoxia-inducible factor (HIF)-1alpha, and bcl-2 in benign and dysplastic cervical squamous epithelia and invasive squamous cell carcinomas (ISCCs). EpoR expression in benign epithelia was confined to the basal cell layers, whereas in dysplasias it increasingly appeared in more superficial cell layers and showed a significant correlation with severity of dysplasia. Diffuse EpoR expression was found in all ISCCs. Expression of Epo and HIF-1alpha was increased in dysplasias compared to benign epithelia. Focal Epo and HIF-1alpha expression was seen near necrotic areas in ISCCs, and showed correlation in their spatial distribution. Significant correlation was found between expression of EpoR, and p16 and bcl-2 in benign and dysplastic squamous epithelia. Our results suggest that increased expression of Epo and EpoR may play a significant role in cervical carcinogenesis and tumor progression. Hypoxia-inducible Epo signaling may play a significant role in the aggressive behavior and treatment resistance of hypoxic cervical cancers.

Expression of 60-kD Heat Shock Protein Increases during Carcinogenesis in the Uterine Exocervix

Objectives: The aim of the present study was to determine the presence and expression of the 60-kD heat shock protein (HSP60) in the dysplasia-carcinoma sequence in the uterine exocervix and to evaluate its diagnostic and prognostic significance. Methods and Results: We performed Western blot and immunohistochemical analyses on biopsies from 40 cases, consisting of 10 normal exocervical biopsies, 10 low-grade squamous intraepithelial lesions (L-SIL), 10 high-grade squamous intraepithelial lesions (H-SIL) and 10 cancerous exocervices (G2 grade). The immunohistochemical results were quantified by computer-assisted image analysis. Western blot analysis showed that HSP60 was undetectable in normal tissues and that there was a gradual increase of protein expression from L-SIL to carcinoma. Immunostaining for HSP60 was negative in normal tissue and positive in basal and parabasal layers of L-SIL epithelium; H-SIL were markedly stained in all layers of epithelium, and carcinomas showed an even stronger positivity. The increasing expression correlated with the malignancy grade. Finally, koilocytes were mostly negative in L-SIL and positive in H-SIL. Conclusions: The increasing degree of expression of HSP60 from L-SIL to carcinoma and the different intraepithelial distribution between L-SIL and H-SIL could be used as a new diagnostic tool. Moreover, HSP60 could have a role in cervical carcinogenesis.

Improved immunodetection of human papillomavirus E7.

Human papillomavirus E7 (HPV E7) is a viral oncoprotein that plays an important role in cervical carcinogenesis through binding with retinoblastoma protein (Rb). Inactivation of Rb by E7 is necessary but not sufficient for cellular transformation, suggesting other protein-protein interactions are required for E7-mediated cellular transformation aside from the interaction with Rb. However, studies on the oncogenic function of HPV E7 have been limited by its poor immunoreactivity. In this report, we show that the fixation of purified recombinant HPV E7 on blotted nitrocellulose membrane with glutaldehyde markedly enhanced the immunoreactivity of HPV E7 protein. Using HeLa and Caski cell lines which are infected with HPV 18 and HPV 16, respectively, we demonstrated that native HPV E7 proteins also could be detected by this method. These results therefore can provide the experimental conditions for detection of HPV E7 proteins with greater sensitivity and may help to analyze E7 functions.

Herpes simplex virus and risk of cervical cancer: a longitudinal, nested case-control study in the nordic countries.

Human papillomaviruses (HPVs) play the major role in cervical carcinogenesis. The authors reevaluated the role of herpes simplex virus type 2 (HSV-2) in this multistage process by conducting a longitudinal, nested case-control study using 1974-1993 data and comparing the results with those from a meta-analysis of studies. A Nordic cohort of 550,000 women was followed up for an average of 5 years, after which 178 cervical carcinoma cases and 527 controls were identified. HSV-2; HPV-16, HPV-18, and HPV-33; and Chlamydia trachomatis antibodies were determined at baseline by HSV-2 glycoprotein gG-2 and HPV virus-like-particle enzyme immunoassays and by using the microimmunofluorescence method. The relative risk of cervical carcinoma was calculated by conditional logistic regression. Longitudinal studies on HSV-2 and cervical neoplasia were identified through MEDLINE (National Library of Medicine, Bethesda, Maryland), and weighted mean relative risks were calculated. Smoking (relative risk = 1.6, 95% confidence interval (CI): 1.1, 2.3) and HPV-16/HPV-18/HPV-33 (relative risk = 2.9, 95% CI: 1.9, 4.3) were both associated with cervical carcinoma. The smoking- and HPV-16/HPV-18/HPV-33-adjusted relative risks for HSV-2 were 1.0 (95% CI: 0.6, 1.7) and 0.7 (95% CI: 0.3, 1.6), respectively, for HPV seropositives. In the meta-analysis, the relative risk for HSV-2 was 0.9 (95% CI: 0.6, 1.3). In both sets of data, HSV-2 did not play a role in cervical carcinogenesis.

Frequency of herpes simplex virus, cytomegalovirus and human papillomavirus DNA in semen.

Herpes simplex virus (HSV-2) and cytomegalovirus (CMV) infections produce brain damage in the newborn, and human papillomavirus (HPV) plays a role in cervical carcinogenesis. To assess the frequency of herpes virus and HPV in semen and its role in transmission, semen from 111 male partners of women with histologically-detected genital HPV infection was analysed for HSV, CMV and HPV infection. We used cell culture to detect HSV and CMV, and polymerase chain reaction (PCR) for HPV. Virological findings in the sperm were correlated to the presence or absence of HPV-associated genital lesions and to the viral type. Viral cultures yielded HSV-2 DNA in 9% and CMV DNA in 6.3% of cases. No correlation was established with a history of clinically apparent infection for HSV. HPV-DNA was detected in 23.4% of semen by PCR techniques: in 48% of subjects with urethral lesions, in 22% of patients with penile lesions, in 2% of patients without HPV-associated lesions. HPV-DNA type 16 was detected in 3.6% of cases. Patients with a positive HPV semen sample and penile or urethral lesions had the same HPV type detected in the two specimens. The study shows a high detection of clinically inapparent HSV and CMV, but does not confirm high HPV prevalence in semen from men without detectable lesions. Our study also suggests that the mechanism for semen contamination by HPV is the exfoliation of infected cells from urethral lesions during semen ejaculation, and probably, by abrasion from penile lesions. This could result in the contamination of semen used in assisted reproductive technology.

Serum Vascular Endothelial Growth Factor and Serum Leptin in Patients with Cervical Cancer

Objective. Serum levels of vascular endothelial growth factor (VEGF) can be seen as surrogate markers of angiogenesis. Recently, leptin, which is involved in the control of satiety and energy expenditure, was also shown to modulate angiogenesis. As angiogenesis plays an abundant role in cervical carcinogenesis, we evaluated serum VEGF and leptin in patients with cervical intraepithelial neoplasia (CIN) and cervical cancer.Methods. Serum VEGF and leptin were measured in 84 patients with cervical cancer, in 28 patients with CIN I–III, and in 35 healthy women, using a commercially available enzyme-linked immunosorbent assay and radioimmunoassay, respectively.Results. Serum VEGF was significantly elevated in patients with cervical cancer and in patients with CIN I–III compared to healthy women. In patients with cervical cancer serum VEGF was significantly correlated with tumor stage, but not with lymph node involvement and histological grade. Univariate and multivariate analyses showed that elevated pretreatment serum VEGF was not associated with the duration of disease-free and overall survival. Serum leptin did not differ among patients with cervical cancer, patients with CIN I–III, and healthy women. Serum leptin was significantly correlated with body mass index (BMI). All further analyses were performed with absolute and serum leptin corrected by BMI. No differences in serum leptin could be ascertained between patients with cervical cancer and patients with CIN I–III. Serum leptin was not associated with any clinicopathological parameter and patients' survival. No correlation between serum VEGF and leptin was found.Conclusions. It can be speculated that serum VEGF might be used as a surrogate marker of angiogenesis in patients with cervical cancer. Our data support the concept that VEGF plays a role in malignant transformation and tumor growth, but not in the lymphatic spread of cervical cancer. This is the first report on leptin in a gynecological malignancy. Our results show that serum leptin falls short of being a useful marker in patients with cervical cancer.

P53 immunohistochemical expression: messages in cervical carcinogenesis

The pattern of p53 expression was studied in pre-invasive and invasive cervical carcinoma in an attempt to clarify its role in cervical carcinogenesis. A total of 234 invasive cervical carcinomas (152 squamous cell carcinomas, 61 adenocarcinomas and 21 adenosquamous carcinomas) and 16 cervical intraepithelial neoplasia (CIN) I, six CIN II and 25 CIN III were immunohistochemically studied for p53. p53 was detected more frequently in CIN and invasive carcinoma (100% of CIN I, 74.2% CIN II + III and 70.1% invasive carcinoma) compared with benign cervices (P< 0.001); however, only three squamous cell carcinomas, 11 adenocarcinomas and two adenosquamous carcinomas exhibited p53 expression in >75% of tumour nuclei. Six of the 11 adenocarcinomas and both adenosquamous carcinomas were poorly differentiated compared with one of the three squamous carcinomas. p53 immunoreactive cells were randomly distributed in invasive carcinoma, confined to the lower third of the epithelium in CIN I, reached the middle third in 20% of CIN II and upper third in 16.6% of CIN III. Assuming that p53 immunoreactivity indicates gene mutation when the majority (> 75%) of neoplastic cells express p53, p53 mutations would seem uncommon in cervical carcinogenesis. Nonetheless, glandular malignancies, in particular poorly differentiated variants, may show a higher frequency of mutation. p53 was detected more frequently in CIN I compared with CIN II/III and invasive carcinoma which may be due to p53 protein degradation following interaction with high risk human papillomavirus E6 protein in CIN II/III and invasive carcinoma.

8-Hydroxy-2′-deoxyguanosine in cervical cells: correlation with grade of dysplasia and human papillomavirus infection

In this study, the 8-hydroxy-2'-deoxyguanosine (8-OHdG) level was assessed in human cervical cells by an immunoperoxidase method and was related to the presence of human papillomavirus (HPV) infection and precancerous lesions. After optimizing the immunohistochemical method of detecting oxidative DNA damage in whole cells, we have used this technique to estimate the oxidative damage in cervical cells collected during a routine PAP test. The analysis of variance (ANOVA) of the data from human samples showed significant differences in the 8-OHdG content among normal, low-grade and high-grade squamous intraepithelial lesion (SIL, HGSIL and LGSIL, respectively; P < 0.001). In the comparison of the three groups, statistically significant differences were detected between normal SIL and HGSIL (P < 0.001) and between LGSIL and HGSIL (P = 0.003), whereas no statistically significant difference was found between normal SIL and LGSIL (P = 0.1). Grouping observations by HPV status, no significant difference was detected in 8-OHdG levels between HPV(+) and HPV(-) subjects (P = 0.8). The polytomous and proportional odds models, extensions of the logistic regression analysis, showed that the effect of 8-OHdG levels in rising the risk of dysplasia was roughly constant through SIL grades. In conclusion, the immunoperoxidase method, applied to single human cervical cells, provides clear evidence that significant differences exist in 8-OHdG content between normal and dysplastic cells and that oxidative DNA damage might play an important role in cervical carcinogenesis.

Role of TSG101 in Uterine Cervix Cancer

Objective. TSG101 was first described as a possible tumor suppressor gene in breast cancer. To determine whether TSG101 might play a role in cervical carcinogenesis, we examined a panel of cervical cancer cell lines and primary tumor specimens for transcript abnormalities and mutations in TSG101.Methods. Total RNA was derived from cell line cultures or primary tumor specimens. We performed nested reverse transcription polymerase chain reaction (RT-PCR) with eight overlapping primer sets, followed by single-strand conformational polymorphism (SSCP) analysis, to screen for mutations in the TSG101 open reading frame. Representative normal and shifted SSCP bands were sequenced. To identify abnormal-sized transcripts, we performed RT-PCR with primers flanking the open reading frame followed by gel electrophoresis.Results. Mutational analysis was performed on cDNAs from 20 primary cervical tumors and 8 cervical carcinoma cell lines. Two polymorphisms were identified, neither of which resulted in an altered amino acid sequence. Transcript analysis was performed on a subset of 16 primary cervix tumors and 6 cervix carcinoma cell lines. The wild-type transcript (1228 bp) was the dominant transcript expressed in all samples. A transcript measuring 330 bp was detected in 5 of 6 cell lines and 11 of 16 primary tumor specimens.Conclusion. Our results suggest that mutations in TSG101 rarely occur in carcinomas of the uterine cervix. However, the presence of minor aberrant TSG101 transcripts is a common feature. The relationship between aberrant transcription and carcinogenesis should be further investigated.

Fluorescence in Situ Hybridization in Cervical Smears: Detection of Numerical Aberrations of Chromosomes 7, 3, and X and Relationship to HPV Infection

Background. Human papillomavirus (HPV) is known to play a pivotal role in cervical carcinogenesis. Chromosomal aberrations are known to be related to different biological behaviors of malignant lesions. We analyzed whether numerical chromosomal aberrations, related to more aggressive tumor types, are found not only in high-grade squamous intraepithelial lesions (HSIL) but also in low-grade SIL (LSIL) of the cervix and evaluated their relationship to HPV infection.Methods. Eighty women (19 to 74 years of age) were included in this study and grouped according to the Bethesda System: within normal limits (WNL), LSIL, and HSIL. By FISH, chromosomes 7 and X, and in part chromosome 3, were analyzed for numerical aberrations. Using the hybrid capture system HPV detection was performed.Results. All 20 patients with cervical smear WNL had regular diploid chromosomal pattern and were negative for HPV. Thirteen of the 29 (41.2%) patients with LSIL showed trisomy 7, in association with trisomy X in 4 cases (12.9%). Single trisomy X was detected in 4 cases (12.9%). In 3 of 15 (20%) cases analyzed for chromosome 3 trisomy was observed. Trisomy 3 was associated with trisomy 7 and X or with trisomy 7 alone. The hybrid capture test was performed in 16 patients of this group. Two patients were positive for HPV probe A, 9 for probe B, and 2 for A and B, and 3 patients were negative. Twenty-three of the 29 patients (79.3%) with HSIL showed trisomy 7. Twelve of the 29 patients (41.3%) had an additional trisomy X. Single trisomy X was seen in only 2 cases (6.9%). Twenty-two patients with HSIL were tested also for chromosome 3. Nine of the 22 patients (40.9%) showed trisomy 3, associated with trisomy 7 or with trisomy 7 and X. In 25 of the 29 patients HPV detection by the hybrid capture system was performed. HPV probe B was positive in 15 cases (60%). One patient was positive for both probes, A and B. Nine (36%) of the patients with HSIL were negative for both HPV probes. No positivity was observed for HPV probe A alone.Conclusion. Our data confirm the pivotal role of HPV in cervical carcinogenesis as it seems to cause changes in the chromosomal pattern of premalignant lesions. Additionally, trisomy 7 may be considered an early event in cervical carcinogenesis, persisting and increasing with progression of the lesion. The roles of trisomy 3 and X need further evaluation.

Telomerase activity and human papillomavirus (HPV) infection in human uterine cervical cancers and cervical smears

Telomerase activity and human papillomavirus (HPV) infection were investigated in uterine cervical samples using molecular biology techniques. Thirteen cervical carcinomas and corresponding normal tissue from the same patient, and 102 cervical swabs were examined. Telomerase activity was detected in 12 of 13 cervical cancer tissues (92%). Of the 12 cases that showed telomerase activity, all were HPV positive, and the one case that did not show telomerase activity was HPV negative. A telomeric repeat amplification protocol assay detected telomerase activity in one out of seven normal cervical tissues (14%), and this one case was HPV positive. In cervical smear samples, telomerase activity was detected in two out of 36 normal smears (6%; both HPV positive), in 10 of 32 (31%) CIN1 (cervical intraepithelial neoplasia) cases (three HPV positive), in four of five (80%) CIN2 cases (two HPV positive), in 15 of 21 (71%) CIN3 cases, (seven HPV positive) and in seven of eight (88%) squamous cell carcinoma cases (six HPV positive). These results suggest that telomerase activity may play some role in cervical carcinogenesis, and telomerase activity is associated with HPV infection in uterine cervical lesions.

Human papillomavirus infection and risk determinants for squamous intraepithelial lesion and cervical cancer in Japan.

A case control design was used to investigate human papillomavirus (HPV) prevalence and risk factors associated with development of cervical squamous intraepithelial lesion (SIL) and cervical cancer (CC) in Japan, One hundred and twenty‐three women with histologically confirmed SIL or CC were compared to a control group of 778 cytologically normal women. With the use of a polymerase chain reaction (FCR)‐hased method for detection of low‐risk (types 6 and 11) and high‐risk (types 16, 18, 31, 33, 35, 52 and 58) HPVs, a high prevalence of HPV infection was observed in smokers among the controls. Logistic regression analysis demonstrated that high‐risk HPV infection was the most significant risk determinant for LSIL (OR=9.4, 95% CI=4.5–19), HSIL (OR=77, 95% CI=28–217) and CC (OR=97, 95% CI=35–269). It also showed that unmarried women, women married for 5 to 19 years and smokers represented high risk groups for SIL, while smokers and women with a history of many pregnancies/parities had increased risk for CC. Smoking was the only HPV infection‐independent factor for CC, suggesting that smoking may have a carcinogenic effect on the cervix. Since neither history of other cancer nor family cancer history was associated with SIL or CC, genetic factors appear to play little role in cervical carcinogenesis. The risk for cervical neoplasia due to HPV infection increased after marriage in Japan, suggesting a role for husbands as carriers of HPV transmission. Protection from high‐risk HPV infection may be of greatest importance for prevention of cervical cancer.

Interleukin 1alpha and interleukin 6 promote the in vitro growth of both normal and neoplastic human cervical epithelial cells.

Interleukin 1alpha (IL-1alpha), Interleukin 6 (IL-6) and epidermal growth factor (EGF) were tested for their ability to regulate epithelial cervical cell cytokine production and secretion and to induce proliferation of human normal and neoplastic epithelial cervical cells. IL-1alpha, and IL-6 enhanced tumour and normal cell growth by 20-120%. The interleukins efficacy was similar to that of EGF for some cell lines but not for normal esocervical cells. The stimulatory effects of the interleukins were observed in both human papilloma virus (HPV)-infected and HPV-non-infected cervical cells. Normal cells constitutively expressed IL-1alpha, IL-6 and EGF mRNA. All cell lines except C33A expressed IL-1alpha mRNA. CaSki, C-4II and HT-3 expressed mRNA for IL-6. IL-1alpha induced or increased IL-6 mRNA levels in the Me-180 and HT-3 lines and in normal cervical cells. IL-6 induced: (1) the expression of its own mRNA only in Me-180 cells that constitutively lacked IL-6 mRNA; (2) the expression of IL-1alpha mRNA in C-33A and increased IL-1alpha mRNA level in the case of Me180 cells. Increased amounts of IL-6 mRNA were found in normal cells when treated with IL-1alpha. In spite of the pattern of mRNA expression, only HT-3 and normal cervical cells constitutively secreted IL-6, and only normal cells were able to produce IL-1alpha protein. A significant IL-1alpha-dependent increase of IL-6 secretion was found in Me-1 80, HT-3 and normal cells. IL-1alpha- and IL-6-driven cell proliferations were almost completely inhibited by the addition of neutralizing anti-IL-6 antibodies. Taken together, these data suggest that interleukins play a role in cervical carcinogenesis as autocrine and/or paracrine stimuli.