Quantitative real-time polymerase chain reaction analysis of the type distribution, viral load, and physical status of human papillomavirus in liquid-based cytology samples from cervical lesions.

Human papillomavirus (HPV) 16 DNA can be integrated into the DNA of cells, thereby disrupting E2 gene expression, which leads to increased expression of the E6 and E7 viral oncogenes and progression to cancer. However, the relationships among HPV viral load, cytologic diagnosis, and HPV integration status remain unclear. The aim of this study was to evaluate the HPV type distribution, viral load, and HPV 16 integration status, and then investigate their relationships with precancerous and cancerous lesions among Japanese women of different age groups. Liquid-based cytology (LBC) samples were examined by quantitative real-time polymerase chain reaction (PCR). The overall mean prevalences of HPV were higher in younger women and lower in middle-aged women among the age groups. The positivity rate of HPV 16 peaked at a younger age than that of all HPV subtypes. The HPV 16 viral load per cell decreased from a low-grade squamous intraepithelial lesion (LSIL) to a cancerous lesion (257.4 for LSIL, 76.9 for high-grade squamous intraepithelial lesions, and 35.7 for cancerous lesions). The average HPV 16 DNA copy numbers for three different HPV 16 integration statuses were 64.1 for the episomal form, 465.5 for the mixed form, and 0.4 for the integrated form. Furthermore, the mean age of patients with the pure integrated form of HPV 16 was more than 10 years older than those of patients with the episomal and mixed forms. Quantitative real-time PCR appears to be a useful method for quantitative and physical status analyses of HPV in cervical cancer screening with LBC samples.

Sperm protein 17 influences the tissue-specific malignancy of clear cell adenocarcinoma in human epithelial ovarian cancer.

Clear cell adenocarcinoma of the ovary has a poor prognosis due to chemoresistance and early metastasis to the lymph nodes. It also can result in endometriosis and is the second most frequent type of ovarian cancer in Japan. Serous adenocarcinoma of the ovary is another common epithelial cancer tissue subtype in Japan, and it is highly sensitive to chemotherapy. In the current study, we examined the differential expression of genes in these types of ovarian cancer and tried to analyze their functions, especially as they relate to chemoresistance. We used differential display to compare clear cell carcinoma and serous adenocarcinoma of the ovary. We identified sperm protein 17 (SP17) as a candidate gene related to the chemoresistance of clear cell carcinoma. Its differential expression was confirmed by real-time polymerase chain reaction. Because the function of the SP17 gene in ovarian cancer is not known, we examined the effect of small interfering RNA targeting the SP17 gene on the chemoresistance and proliferation of ES-2 ovarian cancer cells to paclitaxel, currently the most effective treatment for ovarian cancer. We found that this treatment decreased the chemoresistance of these cells to paclitaxel. Our results strongly suggest that SP17 plays a role in the resistance of clear cell carcinoma to chemotherapy without influencing their ability to proliferate.

Inhibition of the mammalian target of rapamycin (mTOR) by rapamycin increases chemosensitivity of CaSki cells to paclitaxel.

Paclitaxel, a potent anti-neoplastic agent, has been found to be effective against several tumours, including cervical cancer. However, the exact mechanism underlying the cytotoxic effects of pacitaxel, especially in the survival-signalling pathway, is poorly understood. The aim of this study was to investigate the molecular pathway of the cytotoxic effect of paclitaxel in human cervical cancer cell lines. Four human cervical cancer cell lines were treated for 24 h with various concentration of paclitaxel, and the sensitivity was analysed by an MTT assay. The cell cycle progression and sub-G1 population were analysed by flow cytometry. Apoptosis was further measured by DNA fragmentation and microscope examination. The protein expression was determined by Western blot analysis. Our results showed that HeLa cells demonstrated the highest sensitivity to paclitaxel, whereas CaSki cells showed the lowest. In cervical cancer cells, paclitaxel induced apoptosis through an intrinsic pathway with prior G2/M arrest. In addition, we showed that paclitaxel downregulated the phosphorylation of Akt in both HeLa and CaSki cells. Interestingly, in CaSki cells, which were more suggestive of a resistant phenotype, paclitaxel induced the activation of mTOR as a downstream target of Akt. Pre-treatment with rapamycin inhibited activation of mTOR signalling and significantly enhanced the sensitivity of CaSki cells to paclitaxel by increasing apoptotic cell death. This effect was mediated, at least partly, through caspase activation. Overall, paclitaxel exerts its anti-tumour effects on cervical cancer cells by inducing apoptosis through intrinsic pathway, and rapamycin targeted to mTOR can sensitise paclitaxel-resistant cervical cancer cells.

A case of rhabdomyosarcoma of the vagina in an elderly woman.

Most rhabdomyosarcomas of the vagina (RMSV) occur in infants and children up to six years old. RMSV in elderly patients is extremely rare. We report a case of a 70-year-old woman with RMSV. She had received surgery for uterine endometrial cancer one year before and a vaginal polypoid tumor was noted during routine follow-up vaginal examination. She was referred to our department for radiation therapy following partial tumorectomy of the lesion. She was given three sessions of intra-vaginal radiation therapy, once a week with 6 Gy at 7.5 mm below the vaginal surface and external irradiation of 50 Gy to the pelvis. However, paraaortal lymph node metastasis developed during initial radiation therapy. Furthermore, multiple bone metastases appeared at the completion of the radiation therapy. Six months after initial treatment the patient died from progression of the disease. Autopsy demonstrated small residual tumor at the primary site as well as multiple systemic metastases.

Oestrogen receptor beta expression and depth of myometrial invasion in human endometrial cancer.

We assessed the relative expression of oestrogen receptor (ER)alpha and oestrogen receptor (ER)beta mRNAs in 36 human endometrial cancers using a multiplex polymerase chain reaction (PCR). To determine whether or not the expression of ER subtypes in endometrial cancers is associated with clinicopathological parameters, we examined correlations between ER subtypes and age, tumour grade and depth of myometrial invasion. Using multiple regression analysis, myometrial invasion showed a significant correlation with ER-beta: ER-alpha ratio (r = 0.54, P = 0.0007). The ER-beta:ER-alpha ratio was high in advanced invasive carcinoma. Western blotting analysis showed that ER-beta proteins were highly expressed in comparison with ER-alpha proteins in endometrial cancer with severe myometrial invasion. Our results suggest that ER-beta is important in the progression of myometrial invasion.

Mutation analysis of the Smad6 and Smad7 gene in human ovarian cancers.

The Smad6 and Smad7 genes are members of the Smad family, involved in the transforming growth factor-beta (TGF-beta) signaling pathway. Mutations in TGF-beta receptors and their cytoplasmic elements of transduction signals commonly accompany various cancers. Using PCR-SSCP analysis we searched for the presence of Smad6 and Smad7 gene mutations in 30 human ovarian cancers and 4 ovarian cancer cell lines, and found that 12 cases (35.3%) had a polymorphism in intron 2 of the Smad6 gene and that 8 cases (23.5%) had a polymorphism at codon 208 in the Smad7 gene. Because these polymorphisms were not accompanied by amino acid substitution, the present results show that the mutations in the Smad6 and Smad7 genes are unlikely to be involved in human ovarian cancers.

Analysis of specific gene mutations in the transforming growth factor-beta signal transduction pathway in human ovarian cancer.

Several proteins, including transforming growth factor beta (TGF-beta) receptor type I (RI), TGF-beta receptor type II (RII), Smad2, Smad3, and Smad4/DPC4, have been identified in the transduction pathway of the tumor suppressor TGF-beta. Mutations in TGF-beta RI, TGF-beta RII, Smad2, and Smad4/DPC4 genes are associated with several human cancers. The present study examines these gene mutations in 32 human ovarian cancers and 14 patient-matched normal tissues. For the first time, mutations in the Smad2 and Smad4 genes were analyzed in relation to human ovarian cancer. Gene mutations of TGF-beta RI, TGF-beta RII, Smad2, and Smad4 were analyzed using specific primers by PCR-single-strand conformational polymorphism (SSCP), and the results revealed a frameshift mutation at codons 276-277 (CTCTGG-->CTGCGTGG) in exon 5 of TGF-beta RI in 10 of 32 tumor samples (31.3%). This mutation was associated with reduced or absent expression of TGF-beta RI protein and p53 protein in tumor tissues. We detected SSCP variants of TGF-beta RII in exon 2 in 20 of 32 tumors. Sequence analysis of these variants revealed an A to G transition at the seventh band of intron 2. In this A to G polymorphism in intron 2, 12 samples (37.5%) had A/A alleles, 12 (37.5%) had A/G alleles, and 8 (25%) had G/G alleles. We detected Smad2 SSCP variants in exon 4 in 12 of 32 tumors (37.5%). Sequence analysis revealed a 2-bp deletion in the polypyrimidine tract of intron 3, which is located at position -39 to -56 in the splice acceptor site of the intron 3-exon 4 junction. No SSCP variants were detected in the Smad4 gene. These findings suggest that mutations in the TGF-beta RI and in its signal transduction pathway are likely responsible for human ovarian carcinogenesis.

Mutation analysis of the hMTH1 gene in sporadic human ovarian cancer.

8-oxo-deoxyguanosine triphosphate (8-oxo-dGTP) is a major oxidation product in the nucleotide pool of the cell and is a potent mutagen, because it can be incorporated into DNA with equal frequency opposite either template C or A. The human MTH1 gene (hMTH1) is a homologue of the E. coli mutator gene mutT, which encodes 8-oxo-dGTPase. hMTH1 protein reduces spontaneous mutations by removing 8-oxo-dGTP from the triphosphate pool. To determine whether this gene is associated with carcinogenesis of human ovarian cancer, the present study examined, for the first time, the hMTH1 sequence in 49 ovarian cancers and 9 ovarian cancer cell lines by means of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing analyses. A Gright curved arrow A transition at codon 83 was detected in one patient and one cell line (3.4%), followed by an amino acid change (valineright curved arrow methionine) which was known to cause the protein to be less active in vitro. This one base substitution was found in normal and corresponding tumor DNA, and its allele type was heterozygous. The same change has been detected in HNPCC (hereditary non-polyposis colorectal cancer) and gastric cancer patients, and thus it may not represent a mutation specific for ovarian cancer. A silent Tright curved arrow C transition at codon 119 was detected in 12 patients and 2 cell lines (24.1%). No specific mutations in hMTH1 were found in either ovarian cancer patients or cell lines. Thus, it appears that hMTH1 is not directly associated with ovarian cancer.

Effect of 1,25(OH)2D3 on expression of estrogen receptor-alpha mRNA on rat osteosarcoma cell line (ROS 17/2.8).

In order to investigate the regulatory mechanisms of estrogen receptors (ER) in bone cells, changes in ER-alpha mRNA levels of rat osteosarcoma cell line (ROS 17/2.8) before and after exposure to 1,25(OH)2D3 and 17-beta estradiol respectively were measured by quantitative polymerase chain reaction using an internal standard. ER mRNA levels in the ROS 17/2.8 cultured with the medium alone had 5.029 +/- 1.623 mol/g total RNA x 10(-13) and were not statistically different from those cultured in the presence of 1,25(OH)2D3 at concentrations of 10(-12) M and less. ER mRNA levels in the ROS 17/2.8 cell line showed a small but a significant increase as a result of stimulation by 1,25(OH)2D3 at concentrations of 10(-10) and 10(-11) M. However, ER mRNA levels in ROS 17/2.8 cultured in the presence of 1,25(OH)2D3 at concentrations of 10(-9) M were not statistically different from those of the control. On the other hand, the expression of ER in ROS 17/2.8 cells cultured for 3 hours with various doses of 1,25(OH)2D3 showed, by immunoblotting methods, a significant increase at the dose of 10(-10) M in the expression of ER. Although a physiological significance is obscure, these observations suggest that 1,25(OH)2D3 plays a part in the expression of ER in ROS 17/2.8. No significant changes were seen in the expression of ER mRNA and the synthesis of ER as a result of stimulation by the estradiol.

Mutation analysis of the Smad3 gene in human ovarian cancers.

The Smad3 gene is a member of the Smad family, vertebrate homologues of Drosophila Mad, and its gene product is a cytoplasmic element in the transforming growth factor-beta (TGF-beta) signaling pathway. Mutations in TGF-beta receptors and their cytoplasmic elements of transduction signals commonly accompany various cancers. Using PCR-SSCP analysis we searched for the presence of Smad3 gene mutations in 36 human ovarian cancers, and found that 15 cases (41. 7%) had a polymorphism at codon 103. Because this mutation was not accompanied by amino acid replacement, the present results show that the mutations in the Smad3 gene are unlikely to be involved in human ovarian cancers.

Alterations of the p16INK4A gene in human ovarian cancers.

The p16INK4A gene, which encodes the cell-cycle regulatory protein cyclin-dependent kinase 4 inhibitor, is a putative tumor-suppressor gene. We examined p16 gene alterations in 30 primary ovarian cancers and 11 ovarian cancer cell lines. Five of the primary cancers (16.7%) had lost both p16INK4A genes. In addition, four cancers (13.3%) contained five kinds of missense mutations and a one-base deletion. Three cell lines had homozygous deletions of p16 genes, and one cell line had multiple intragenic mutations. There was also suppressed transcription of the p16 gene in one cell line. Some point mutations occurred in the conserved ankylin consensus region. These observations suggest that p16 is a functional target for ovarian carcinogenesis and that p16 alterations occurred in the primary cancers.

[Chemotherapy and endocrine therapy of endometrial carcinoma].

The main treatment of recurrent and advanced endometrial carcinoma are chemotherapy and/or endocrine therapy. Some of the combination chemotherapy, including Cis-platinum are useful in advanced and recurrent endometrial carcinoma, and progestational agents are available for adjuvant chemo-endocrine therapy after operation of endometrial carcinoma. Authors consider the literatures of the chemotherapy and endocrine therapy for endometrial carcinoma and describe our own cases.

Human leukocyte antigen associated with endometrial carcinoma with a new classification of endometrial carcinoma based on its etiology.

To elucidate the relationship between endometrial carcinoma and the constitution, HLA antigen typing (A-locus, 13; B-locus, 20; C-locus, 6; DR-locus, 9) was investigated in 74 patients with endometrial carcinoma. A significant increase in two HLA antigens, Cw7 and DRw8, was demonstrated, but there was no intimate correlation between Cw7 or DRw8 and the three complications commonly associated with endometrial carcinoma--diabetes mellitus, obesity, and hypertensive disease. On the basis of these results, a new classification of endometrial carcinoma was proposed as follows: type A is positive Cw7 or DRw8 group; type B is negative for Cw7 and DRw8 and positive for the complications mentioned; type C is negative for Cw7 and DRw8, negative for the complications mentioned, and positive for DR 5; type D is a non-A, non-B, and non-C type.