Yoshifumi Nakao

Induction of p16 during immortalization by HPV 16 and 18 and not during malignant transformation.

The p16 (MTS1) tumour-suppressor gene is a cyclin-dependent kinase (cdk) inhibitor that decelerates the cell cycle by inactivating the cdks that phosphorylate the retinoblastoma tumour-suppressor gene (Rb) protein (pRb). In cervical cancers, pRb is inactivated by the HPV E7 oncoprotein or by mutations. The hypothesis of earlier reports was that the disruption of the p16/cdk-cyclin/Rb cascade is essential for malignant cervical transformation/carcinogenesis. We previously established in vitro model systems of cervical cancer representing four steps of oncogenic progression initiated by the two most common oncogenic HPVs in ectocervical and endocervical epithelial cells. This report used these systems to investigate the role of p16 in cervical cancers. A dramatic enhancement of the p16 RNA level was observed after immortalization by HPV 16 or 18. Furthermore, the p16 protein was newly observed following immortalization. However, no further changes were found for RNA or protein levels after serum selection or malignant transformation. For three cervical carcinoma cell lines, similar high levels of p16 expression were seen. Point mutations or homozygous deletions of p16 were not observed in the in vitro systems or in clinical specimens. These results suggest that the inactivation of the p16/cdk-cyclin/Rb cascade does not occur during malignant transformation but occurs during the immortalization by HPV in HPV-harbouring premalignant lesions, the in situ equivalent of immortalized cells. Also suggested is that p16 has no role in the specific malignant transformation step from immortal premalignant lesions during the carcinogenesis of HPV-initiated cervical cancers.

A comparative analysis of human papillomavirus types 16 and 18 and expression of p53 gene and Ki-67 in cervical, vaginal, and vulvar carcinomas

UNLABELLED This study aimed to investigate the correlation between HPV positivity, p53 overexpression, and cell proliferative activity in cervical, vaginal, and vulvar squamous cell carcinoma. METHODS Sixteen vaginal and 31 vulvar squamous cell carcinomas were examined retrospectively for overexpression of p53 gene and Ki67 antigen by immunohistochemistry and for the presence of HPV types 16 and 18 DNA using a polymerase chain reaction (PCR) method. The results were compared with those obtained from 40 cervical squamous cell carcinomas. RESULTS HPV type 16 or 18 DNA was detected in 21 (52.8%) of 40 cases of cervical carcinomas and p53 overexpression in one (2.5%), while HPV DNA sequences were detected in seven (43.7%) of 16 cases of vaginal carcinoma and p53 overexpression in three (18.7%). With regard to vulvar carcinoma, HPV was harbored in four (12.8%) of 31 cases and p53 overexpression in 19 (61.2%). These results indicated statistically significant inverse correlations between HPV positivity and p53 overexpression (R = -0.999, P < 0.0001). Overexpression of Ki-67 was detected in 28 (70.0%) of 40, 12 (75.0%) of 16, and 21 (67.7%) of 31, cervical, vaginal, and vulvar carcinomas, respectively. There was no significant difference among the three groups. CONCLUSIONS In cervical carcinoma, HPV types 16 and 18 might play a common causal role, and in vulvar carcinoma, p53 gene mutations might be a main causal factor for carcinogenesis. Vaginal carcinoma, on the other hand, is considered to have transitional characteristics between cervical and vulvar carcinoma.

Malignant transformation of HPV 16-immortalized human endocervical cells by cigarette smoke condensate and characterization of multistage carcinogenesis.

A number of epidemiological studies indicate that cigarette smokers are at increased risk of developing cervical cancer. However, convincing biological evidence is lacking. This report examines the biological and cellular role of human papillomavirus (HPV) type 16 and cigarette smoke in multistage cervical carcinogenesis. Two lines of HPV16‐immortalized human endocervical cells (HEN‐16 and HEN‐16‐2) generated from primary cells (HEN) were treated with cigarette smoke condensate (CSC). CSC‐treated, but not untreated, HEN‐16 and HEN‐16‐2 formed tumors that were invasive squamous cell carcinomas in nude mice. The tumors were used to initiate 2 tumor lines of cells (HEN‐16T and HEN‐16‐2T, respectively). Cells of both tumor lines, compared with HEN, HEN‐16 and HEN‐16‐2, featured: (a) tumorigenicity, (b) distinct morphologies in monolayer and organotypic (raft) cultures, (c) faster growth in serum plus high calcium levels after immortalization and after transformation, (d) higher saturation density and (e) anchorage‐independent growth. Our results provide unique direct in vitro evidence that cigarette smoke causes cancer in HPV‐containing cervices.

Telomerase Activation in Cervical Neoplasia

Objective To examine the critical point at which telomerase activation occurs in the course of cervical carcinogenesis. Methods Telomeric repeat assay protocol was used to measure telomerase activity in cell samples obtained from 155 Japanese women with various cervical conditions: normal cytology (n = 62), cervical intraepithelial neoplasia (CIN) (n = 63), and invasive squamous cell carcinoma (n = 30). results Telomerase activity was detected in five (8%) women with normal cytology, in 26 (41%) patients with CIN (26% of patients with CIN I, 35% with CIN II, and 68% with CIN III), and in 29 (97%) patients with invasive carcinoma. Telomerase activation was significantly more frequent in CIN than in normal cervices (P < .001), and the positive rate in CIN III was significantly higher than that in CIN I (P < .01) and CIN II (P < .05). Furthermore, telomerase activation was significantly more frequent in invasive carcinoma than in CIN III (P < .01). Conclusion Our findings suggest that telomerase activation is a relatively early event in cervical carcinogenesis and correlates well with grade of cervical lesion.

Successful Pregnancies in 2 Infertile Patients with Endometrial Adenocarcinoma

Two infertile patients with well-differentiated endometrial adenocarcinoma succeeded in having their own babies with assisted reproductive technology following treatment with a high dose of medroxyprogesterone acetate and repeated endometrial curettages. Their follow-up pathological examinations revealed no evidence of recurrent disease. Consequently, conservative treatment may be indicated in patients with well-differentiated endometrial adenocarcinoma at an early stage who desire to preserve their fertility.

Uterine Artery Embolization Followed by Dilation and Curettage for Cervical Pregnancy

BACKGROUND: Cervical pregnancy can be a life-threatening condition due to the risk of severe hemorrhage. Progression of ultrasonographic diagnostic technology has allowed the early detection of cervical pregnancy. However, a standard treatment protocol for fertility preservation has not yet been established. CASE: Two women with cervical pregnancy presented with cardiac activity at 6 and 7 weeks of gestation. They were treated with transfemoral uterine artery embolization followed by dilation and curettage with minimal bleeding. One patient gave birth to a healthy neonate 20 months after the procedure. CONCLUSION: Early cervical pregnancies were treated with dilation and curettage after uterine artery embolization. This treatment can be considered as conservative management for patients who desire to preserve their fertility.

Expression of cellular genes in HPV16-immortalized and cigarette smoke condensate-transformed human endocervical cells.

We studied the molecular mechanism of successive multistep cervical carcinogenic progression with our previously established in vitro model system. This system was composed of primary human endocervical cells (HEN), two lines of HEN immortalized by HPV16 and their counterparts subsequently malignantly transformed by cigarette smoke condensate (CSC). The expression was examined of diverse cellular genes associated with oncogenesis and senescence, especially for cervical cancer. Consistent results were seen for the pairs of immortalized and malignantly transformed lines. Immortalization of HEN by HPV16 resulted in enhanced expression of H‐ras, c‐myc, B‐myb, p53, p16INK4 and PCNA mRNA; enhanced expression of p16 and PCNA proteins; decreased expression of WAF1/p21/Cip1/Sid1 and fibronectin mRNA; and decreased p53 protein. On the other hand, the CSC‐transformed counterparts of HPV16‐immortalized cells had up‐regulated levels of B‐myb, p53 and WAF1 mRNA and p53 protein. Our results indicate that the differential activation or inactivation of multiple cellular genes is important for the immortalization, as well as the transformation, of human cervical cells. Further, we suggest that our in vitro model system is useful for investigating the molecular mechanism of multistep cervical carcinogenesis. J. Cell. Biochem. 66: 309–321, 1997.

The usefulness of combined measurements of squamous cell carcinoma antigens 1 and 2 in diagnosing atopic dermatitis

Background The squamous cell carcinoma antigen (SCCA) is widely used as a serological biomarker for various cancers. There are two known SCCA molecules, SCCA1 and SCCA2. We previously found that interleukin-4 or interleukin-13, two related Th2-type cytokines that play an important role in allergic diseases, induce expression of SCCA1 and SCCA2. In this study, we examined whether combined measurements of SCCA1 and SCCA2 are useful for diagnosing atopic dermatitis (AD). Methods We established new enzyme-linked immunosorbent assays (ELISAs) to specifically detect SCCA1 or SCCA2. We applied serum samples from AD patients with food allergies and from cervical cancer patients to these ELISAs. We performed receiver operating characteristic analyses to diagnose AD and to distinguish AD from cervical cancer. Results Serum concentrations of both SCCA1 and SCCA2 were elevated in AD patients. The serum concentrations of SCCA1 and SCCA2 positively correlated with the clinical severity of AD, showing high specificity (0.86–0.88) and sensitivity (0.86) against control donors. The serum concentrations of SCCA1 and SCCA2 were elevated in cervical cancer patients; however, the SCCA2/SCCA1 ratios clearly distinguished AD patients from cervical cancer patients with high specificity (0.87) and sensitivity (0.87). Expression of SCCA2 was predominant in AD patients, whereas cervical cancer patients showed a predominance of SCCA1. Conclusions Combined measurements of SCCA1 and SCCA2 are very useful in estimating the severity of allergic diseases, making it possible to distinguish allergic diseases from cancers.

Retinoic acid and interferon-α effects on cell growth and differentiation in cervical carcinoma cell lines

OBJECTIVE To investigate and compare the efficacy of all‐trans retinoic acid (RA) and/or interferon‐α (IFN‐α) on premalignant and malignant models of cervical cancer. METHODS Cell growth rate was examined after treatment for 4, 7, and 10 days with RA and/or IFN‐α of human papillomavirus type 18 (HPV 18)‐immortalized endo‐ and ectocervical cells, nontransformed serum‐adapted cells, transformed cells, three adenocarcinoma, and three squamous cell carcinoma cell lines. The effect on epithelial differentiation by RA and IFN‐α was examined in organotypic culture. Induction of apoptosis was examined by modified terminal transferase‐mediated deoxyuridine triphosphate‐biotin nick end‐labeling (TUNEL) and DNA fragmentation. RESULTS Cell growth rate was inhibited by RA, 84–96% in HPV 18‐immortalized endocervical cells, SiHa, and ME180, 0% in OMC‐4, and 18–62% in other cell lines; and by IFN‐α about 75% in SiHa and ME180 and 14–40% in the other cell lines. Combining RA and IFN‐α increased the antiproliferative effect in premalignant cell lines and some cancer cell lines except OMC‐4, SiHa, and HT‐3. In rafts, RA treatment reversed human endocervical cell metaplasia and HPV 18‐immortalized endo‐ and ectocervical cell dysplastic epithelial differentiation. Interferon‐α, not RA, treatment of HPV 18‐immortalized endo‐ and ectocervical cells induced apoptosis. CONCLUSION Cell growth inhibition by treatment with RA, IFN‐α, and their combination differentially depends on treatment type and time, cell origin, cell line, and oncogenic state. In a premalignant model of cervical carcinoma, RA reduces dysplastic differentiation and IFN‐α induces apoptosis. These data confirm that these treatments may be effective for preventing or treating premalignant cervical lesions.

Alterations in physical state and expression of human papillomavirus type 18 DNA following crisis and establishment of immortalized ectocervical cells.

Integration of episomal human papillomavirus (HPV) DNA in infected cervical lesions during malignant progression is frequently observed, but the importance of integration is poorly understood. We have studied immortalization by HPV-18 of human cervical cells as an in vitro model system. Here, the status and expression of HPV-18 DNA in precrisis ectocervical keratinocytes was compared with that in the same cells after crisis and establishment of immortalization. Southern blots revealed, and two-dimensional gel analysis confirmed, that the precrisis culture contained more than 100 copies/cell of episomal HPV-18 DNA and no detectable integrated viral DNA. In contrast, the postcrisis cells contained a low copy number of only integrated viral genome. The Northern blot patterns of E6-E7 and E2/E4 RNA expression were also different. Analysis of RNA by RT-PCR indicated that neither culture expressed the unspliced HPV-18 E6 oncogene present in tumor cell lines and that the precrisis, but not postcrisis, culture expressed the full-length E2 repressor. The two cultures displayed a similar keratinocyte morphology in vitro and a similar low grade dysplasia in vivo and both were non-tumorigenic. These results suggest that, although insufficient for complete malignant conversion, viral DNA integration during crisis is associated with the establishment of an immortalized phenotype in which HPV-18 DNA is integrated and HPV-18 RNA expression is altered.