Katsumi Tsukazaki

p16INK4a overexpression and human papillomavirus infection in small cell carcinoma of the uterine cervix

Carcinogenesis of cervical cancer has been investigated, and p16(INK4a) overexpression in squamous cell carcinoma of the cervix has been reported as a result of infection by human papillomavirus (HPV) (eg, HPV 16), and the consequence of the retinoblastoma (Rb) protein inactivation by HPV E7 protein. However, to our knowledge, there have been no studies on the relation between p16(INK4a) overexpression associated with HPV and small cell carcinoma of the cervix, which behaves more aggressively clinically than squamous cell carcinoma. The purpose of this study was to determine whether p16(INK4a) is overexpressed in small cell carcinoma, and if p16(INK4a) is overexpressed, the types of HPV that are related to this cancer. We reviewed 10 cases of small cell carcinoma and examined them for p16(INK4a) overexpression by immunohistochemistry. We also performed HPV typing with polymerase chain reaction (PCR)-sequencing analysis and in situ hybridization and found that p16(INK4a) was overexpressed in every case. PCR-sequencing analyses revealed that all cases were HPV-positive and that 9 cases were positive for HPV 18. Five of the 9 cases positive for HPV 18 were also positive by in situ hybridization and yielded a punctate signal, considered to represent the integrated form. In conclusion, p16(INK4a) was overexpressed and HPV 18 was frequently detected in an integrated form in small cell carcinoma. Therefore, inactivation of Rb protein by HPV 18 E7 protein may be associated with carcinogenesis of small cell carcinoma the same as inactivation of Rb protein by HPV 16 E7 protein is associated with carcinogenesis of squamous cell carcinoma.

Correlation of p16INK4A overexpression with human papillomavirus infection in cervical adenocarcinomas.

As human papillomavirus (HPV) infection is the main risk factor for squamous cell carcinoma of the cervix and overexpression of p16INK4a occurs when retinoblastoma protein is inactivated by high-risk HPV, the authors studied the association of HPV infection and expression of p16INK4a in cervical adenocarcinomas. Specimens of cervical glandular neoplasias were immunostained with a p16INK4a-specific monoclonal antibody (clone E6H4). Approximately 80% of glandular neoplasms showed overexpression of p16INK4a. Exfoliated cells from 14 adenocarcinomas were further examined by p16INK4a-specific immunocytochemistry, and 12 cases showed overexpression of p16INK4a, suggesting that immunostaining for p16INK4a may be a useful diagnostic tool for cervical adenocarcinomas. The authors further examined HPV DNA in cervical adenocarcinomas with the polymerase chain reaction method. Overexpression of p16INK4a was positive in 94% of cases in which HPV16 or 18DNA was positive, a finding suggesting that HPV16 or 18 may play an important role in cervical adenocarcinomas. Overexpression of p16INK4a may be an indicator of pathogenic activity of high-risk HPVs.

Alterations in the glycolipid composition and cellular properties of ovarian carcinoma-derived RMG-1 cells on transfection of the α1,2-fucosyltransferase gene

Transfection of the mouse Fut1 and Fut2, and human FUT1 genes into human ovarian carcinoma‐derived RMG‐1 cells resulted in 20–30‐fold increases in cellular α1,2‐fucosyltransferase activity, and in alteration of the glycolipid composition, including not only fucosylated products, but also precursor glycolipids. Although globo‐series glycolipids were not significantly affected by the transfection, the major glycolipids belonging to the lacto‐series type 1 chain family in RMG‐1 cells and the transfectants were the Lc4Cer, Lewis a (Le)a and Leb, and H‐1 glycolipids, respectively, suggesting that fucosylation of Lc4Cer to the H‐1 glycolipid prevents the further modification of Lc4Cer to Lea and Leb in the transfectants. Also, the lacto‐series type 2 chains in RMG‐1 cells were LeX, NeuAc‐nLc4Cer and NeuAc‐LeX, and those in the transfectants were LeX and LeY, indicating that the sialylation of nLc4Cer and LeX is restricted by increased fucosylation of LeX. As a result, the amount of sialic acid released by sialidase from the transfectants decreased to 70% of that from RMG‐1 cells, and several membrane‐mediated phenomena, such as the cell‐to‐cell interaction between cancer cells and mesothelial cells, and the cell viability in the presence of an anticancer drug, 5‐fluorouracil, for the transfectants was found to be increased in comparison to that for RMG‐1 cells. These findings indicate that cell surface carbohydrates are involved in the biological properties, including cell‐to‐cell adhesion and drug resistance, of cancer cells. (Cancer Sci 2005; 96: 26 –31)

Comparison between in situ hybridization and real-time PCR technique as a means of detecting the integrated form of human papillomavirus 16 in cervical neoplasia

Integration of the human papillomavirus (HPV) genome is thought to be one of the causes of cancer progression. However, there is controversy concerning the physical status of HPV 16 in premalignant cervical lesions, and there have been no reports on the concordance between detection of the integrated form of HPV16 by real-time PCR and by in situ hybridization. We investigated specimens of cervical intraepithelial neoplasia (CIN) and invasive carcinomas for the physical status of HPV 16 by real-time PCR and in situ hybridization. The presence of the integrated form was detected by both real-time PCR and in situ hybridization in zero of four cases of CIN1, three of six cases of CIN2, nine of 27 cases of CIN3, and two of six cases of invasive carcinomas. Integrated HPV 16 was present in some premalignant lesions but was not always present in carcinomas. The concordance rate between the two methods for the detection of the presence of the integrated form was 37 of 43 (86%) cases. Real-time PCR and in situ hybridization were found to be complementary and convenient techniques for determining the physical status of the HPV genome. We conclude that a combination of both methods is a more reliable means of assessing the physical status of the HPV genome in cervical neoplasia.

Immunofluorescence-detected infiltration of CD4 +FOXP3 + regulatory T cells is relevant to the prognosis of patients with endometrial cancer

Objective Host antitumor immune responses are associated with many types of immune cells and soluble components. In particular, CD8+ cytotoxic T lymphocytes (CTLs) play a central role. Regulatory T cells (Tregs) have been reported to induce tumor immune tolerance in various cancers. In the present study, we evaluated lymphocytic infiltration in endometrial cancer tissue to clarify its relationship with clinicopathological factors and the prognosis of patients. Methods The study included 53 patients whose condition was diagnosed as endometrial cancer between 1994 and 2004 at Keio University hospital. Using formalin-fixed, paraffin-embedded specimens of the uterus, immunohistochemistry was performed with antihuman CD8, antihuman CD4, and antihuman FOXP3 primary antibodies, and the binding sites of the antibodies were visualized using fluorescence-conjugate secondary antibodies. CD4+FOXP3+ cells were identified as Tregs in this study. The numbers of CD8+ cells, CD4+ cells, and Tregs as well as the Treg/CD8+ and Treg/CD4+ ratios were analyzed to evaluate the relationship between clinicopathological factors and patient prognosis. Results Of the 53 patients studied, 50.9% of them had early-stage disease, 49.1% had advanced stage disease, 47.2% had well-differentiated cancer (grade [G] 1), 24.5% had moderately differentiated cancer (G2), and 28.3% had poorly differentiated cancer (G3). The CD8+ and CD4+ cell counts, Treg count, and Treg/CD8+ and Treg/CD4+ ratios were significantly higher in the patients with advanced poorly differentiated carcinomas and with positive lymphovascular space invasion than in those with early well-differentiated carcinomas and with negative lymphovascular space invasion. In disease-free survival, the prognosis of the patients with high Treg counts and Treg/CD8+ ratios was significantly worse than that of the patients with low Treg counts and Treg/CD8+ ratios (P < 0.05). Conclusions The Treg count and Treg/CD8+ ratio may be new prognostic factors for endometrial cancer.

Papanicolaou tests and molecular analyses using new fluid-based specimen collection technology in 3000 Japanese women

A fluid-based Papanicolaou test has been established to improve sample collection and preparation. This study was the first large-scale investigation in Japan to examine the feasibility of using fluid-based Papanicolaou specimens to detect human papillomavirus (HPV) using Hybrid Capture II and polymerase chain reaction (PCR). Three thousand patients who visited Keio University Hospital between October 2000 and February 2001 were enrolled in the study. The results of the fluid-based Papanicolaou tests corresponded well with those of conventional Papanicolaou smears (96.8% concordance). The sensitivities of cervical neoplasia detection using the fluid-based Papanicolaou test (73.9%) and Hybrid Capture II (76.3%, P=0.55) were not significantly different. Among the cervical intraepithelial neoplasia 3 and squamous cell carcinoma specimens, HPV 16 and HPV 52 were predominantly detected using the PCR method. Although some DNA samples extracted from the fluid-based specimens were degradaded, PCR and direct sequencing could be performed without difficulty even after 1 year of specimen storage. We conclude that fluid-based Papanicolaou specimens can be applied to investigate HPV infection.

Estrogen sulfotransferase and sulfatase: Roles in the regulation of estrogen activity in human uterine endometrial carcinomas

The regulation of estrogen activity through the formation and cleavage of sulfoconjugates of estrogens is known to be related to the progression and metastasis of estrogen‐dependent breast carcinomas, but the involvement of sulfoconjugates in the steroid stimulation of endometrial functions and the progression of endometrial adenocarcinomas is not clearly understood yet. Estrogen sulfotransferase (EST) in the uterine endometria during the follicular phase was more active than during the luteal phase, but estrogen sulfate (ES) sulfatase exhibited lower activity during the follicular phase than during the luteal phase. However, ES sulfatase activities in cancerous tissues were lower than those in normal endometria and endometrial adenocarcinoma‐derived cells, among which the activity was exceedingly high in Ishikawa cells, suggesting that ES sulfatase in Ishikawa cells contributes to the estrogen‐dependent growth of these cells. EST activities higher than that in Ishikawa cells were found in only 3 of 24 cancerous tissues. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis of the EST and ES sulfatase genes in carcinoma‐derived cells demonstrated the extensive expression of both genes in Ishikawa cells. The isolated EST gene was transfected into Ishikawa cells with a mammalian expression vector to establish cell clones with enhanced EST activity, and the estrogen‐dependent cell growth of the resultant cell clones was found to be abolished, due to the enhanced sulfoconjugation of estrogen. Since ES sulfatase activity in cancerous tissues was significantly lower than that in Ishikawa cells, it might be not involved in the enhancement of estrogen activity associated with the pathogenesis of endometrial adenocarcinoma tissues.

HMMC-1 A Humanized Monoclonal Antibody With Therapeutic Potential Against Müllerian Duct-Related Carcinomas

Purpose: The purpose of this research was to generate a human monoclonal antibody specific to gynecological cancers and to evaluate such an antibody as therapy for gynecological cancers. Experimental Design: Transchromosomal KM mice were immunized with the human uterine endometrial cancer cell line SNG-S. Hybridomas were constructed between spleen cells from KM mice and mouse myeloma cells. Reactivity of the antibody was evaluated by immunohistochemistry of pathological specimens of gynecological cancers. Cytotoxicity of HMMC-1 against SNG-S cells was tested by in vitro cytotoxicity assays. The epitope of HMMC-1 was determined by transfection with a panel of glycosyltransferase cDNAs and by inhibition assays with chemically synthesized oligosaccharides. Results: HMMC-1 is a human IgM monoclonal antibody that reacts positively with müllerian duct-related carcinomas with positive rates of 54.6% against uterine endometrial adenocarcinoma, 76.9% against uterine cervical adenocarcinoma, and 75.0% against epithelial ovarian cancer. HMMC-1 does not react with normal endometrium at proliferative or secretory phases, normal uterine cervix, or normal and malignant tissue from other organs, whereas it reacts weakly with the epithelium of the gall bladder and the collecting duct of the kidney. HMMC-1 exhibits antigen-dependent and complement-mediated cytotoxicity. Upon cotransfection with cDNAs encoding two glycosyltransferases required for fucosylated extended core 1 O-glycan, mammalian cells express HMMC-1 antigen. Finally, binding of HMMC-1 to SNG-S cells is inhibited by synthetic Fucα1→2Galβ1→4GlcNAcβ1→3Galβ1→3GalNAcα1-octyl. Conclusions: These results indicate that HMMC-1 specifically recognizes a novel O-glycan structure. The unique specificity and cytotoxicity of HMMC-1 strongly suggest a therapeutic potential of this antibody.

Selection of human ovarian carcinoma cells with high dissemination potential by repeated passage of the cells in vivo into nude mice, and involvement of Le(x)-determinant in the dissemination potential.

Cells of the human tumor cell line RMG‐1, derived from a clear‐cell adenocarcinoma of the ovary, were injected intraperitoneally into nude mice, and the cells obtained from the tumor nodules in the mesenterium were found to form a larger number of, and larger‐sized, tumor nodules than the original RMG‐1 cells. The RMG‐1‐h cells, transferred into culture from the tumor nodules after a 4th in vivo passage, showed a dissemination potential as high as that of cells disseminating directly from the tissues, and exceedingly higher than that of RMG‐1 cells. To assess the molecular bases of the different biological properties of RMG‐1 and RMG‐1‐h cells, we compared the content and expression of various carbohydrate antigens in both cells. The chromosomal profile of RMG‐1‐h cells revealed their human origin and was identical to that of the original RMG‐1 cells. In contrast to the broad histogram for the Lex‐bearing cells among RMG‐1 cells in flow cytometry, the weakly and moderately positive cells toward anti‐Lex antibody were found to be eliminated from the histogram for the RMG‐1‐h cells, resulting in the enrichment of cells strongly expressing Lex, which may account for the high dissemination potential. In addition, the adhesion of RMG‐1 cells to mesothelial cells was found to be significantly inhibited by pretreatment of the cells with anti‐Lex antibody, indicating Lex‐mediated cell‐to‐cell interaction between ovarian cancer cells and mesothelial cells. By TLC‐immunostaining, two Lex‐glycolipids, III3Fucα‐nLc4Cer and V3Fucα‐nLc6Cer were detected in both RMG‐1 and RMG‐1‐h cells, and their total concentrations were not significantly different from each other. However, the hydrophobic moieties of Lex‐glycolipids in RMG‐1‐h cells were different from those in RMG‐1 cells, suggesting that a difference in the structure of the hydrophobic moieties of Lex is partly involved in the enhanced reactivity of RMG‐1‐h cells toward anti‐Lex antibody. Thus, the high dissemination potential of ovarian cancer cells was shown to be mediated by the Lex‐determinant and the Lex‐bearing cells are enriched by repeated in vivo passage of the cells into nude mice.

Enzymatic basis for the accumulation of Lewisb antigen in uterine endometrial cancer

In order to clarify the mechanism of the abnormal expression of Lewisb antigen, which was specific for uterine endometrial cancer tissue, the activities of α1→2fucosyltransferase, α1→3fucosyltransferase, and α1→4fucosyltransferase in normal endometrial tissues and uterine endometrial cancer tissues were determined. Further, an immunocytochemical study of the expression of blood group‐related carbohydrate antigens in 6 cultured cell lines derived from various gynecologic malignant tumors was performed and the α1→2fucosyltransferase, α1→3fucosyltransferase, and α1→4fucosyltransferase activities of these cell lines were determined. Compared with normal endometrium, uterine endometrial cancer tissues showed significantly higher values of α1→2fucosyltransferase, α1→3fucosyltransferase, and α1→4fucosyltransferase activities. The specifically strong expression of type I carbohydrate chains, particularly the Lewisb antigen, was recognized in cultured cell lines derived from uterine endometrial cancer. Compared with those cell lines derived from uterine cervical cancer and ovarian cancer, the cultured cell lines derived from uterine endometrial cancer showed higher activities of α1→2fucosyltransferase and α1→4fucosyltransferase, which are enzymes related to the synthesis of Lewisb antigen. The cell lines derived from uterine endometrial cancer showed specifically high values of α1→4fucosyltransferase activity. These results suggest that the α1→2fucosyltransferase and α1→4fucosyltransferase activities, especially the α1→4fucosyltransferase activity, contribute to the abnormal expression of the Lewisb antigen in uterine endometrial cancer.