Katsunari Yane

Transfection with mutant p53 gene inhibits heat-induced apoptosis in a head and neck cell line of human squamous cell carcinoma

PURPOSE To confirm that human cancer cells show p53-dependent heat sensitivity through an apoptosis-related mechanism, we examined the heat sensitivity and Bax-mediated apoptosis after heating in a human squamous cell carcinoma cell line, SAS, with identical genetic backgrounds except for the p53 status. MATERIALS AND METHODS We performed colony formation assay, Western blotting and analyses of apoptosis, using the SAS cells transfected with pC53-248 vector with mutant p53 gene (SAS/Trp248 cells) or the cells transfected with pCMV-Neo-Bam vector (SAS/neo cells) as a control. RESULTS SAS/Trp248 cells showed heat resistance due to the dominant negative nature of mp53, compared with SAS/neo cells. The incidence of DNA ladders and apoptotic bodies increased markedly after heating in SAS/neo cells, but increased very little in SAS/Trp248 cells. CONCLUSION These results suggest that heat resistance brought by mp53-transfection is p53-dependent and closely correlates with the induction of apoptosis in human squamous cell carcinomas.

Expression of the estrogen receptor in human thyroid neoplasms

The expression and quantitation of the estrogen receptor (ER) in human thyroid tumors were examined by biochemical, immunohistochemical, and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. For this study, neoplasms, adenomatous goiters and adjacent normal thyroid tissues were obtained from 35 patients which included 10 cases of papillary carcinomas, 17 cases of adenomas and 8 cases of adenomatous goiters. Regardless of the histopathological subtype, ER was detected in 19% (5/27) of the neoplastic tissues with the mean value of ER content of 5.0 +/- 1.3 fmol/mg protein and the mean Kd value of 0.38 +/- 0.28 nM. ER was also detected, but at a lower concentration (2.8 +/- 1.6 fmol/mg protein), in the surrounding normal tissues. There was no significant difference between the neoplasms and adenomatous goiters with respect to the incidence of ER positivity and ER content. Furthermore, ER-positive specimens, as determined by both biochemical and immunohistochemical techniques, also showed the expression of ER mRNA detected by RT-PCR method. These results demonstrate that both ER mRNA as well as ER protein are expressed in thyroid neoplasms. This suggests the possibility that estrogen may affect the tumorigenesis or the progression of some thyroid neoplasms.

Immunohistochemical analysis of estrogen receptors in 313 paraffin section cases of human thyroid tissue

Three hundred and thirteen cases of human thyroid tissues, comprising 39 nodular goiters from 34 females and 5 males, 130 adenomas from 93 females and 37 males, and 144 carcinomas from 99 females and 45 males were used for the present immunohistochemical assessment of estrogen receptor (ER) expression. Thirty-three cases of follicular carcinoma, 115 cases of papillary carcinoma and 6 cases of anaplastic carcinoma were included in the malignant tumor group. Incidences of ER-positive cases were 23/39 (58.9%) for nodular goiter, 44/130 (33.8%) for adenoma and 26/144 (18.0%) for cancer. In the individual carcinoma categories, 7/23 (30.4%) follicular, 19/115 (16.5%) papillary and 0/6 (0%) anaplastic lesions were judged as positive cases. Thus, the incidence of ER-positive cases tended to decrease with the degree of malignancy; this trend being similar in both sexes. Moreover, the average ages of ER-positive cases were lower than those of ER-negative cases for all types of thyroid carcinoma except the follicular variety in males. It was thus suggested that ER expression may be related to prognosis and tumor growth at early stage. Since the incidence of ER does not significantly differ between females and males, the observed sex differences regarding thyroid tumor incidence may reflect the higher estrogen serum content in females.

Transfection of mutant p53 gene depresses X-ray- or CDDP-induced apoptosis in a human squamous cell carcinoma of the head and neck

The present study examined whether X-ray- and CDDP-sensitivities depend on p53 gene status in human squamous cell carcinoma of the head and neck (SAS cells) showing dominant negative nature of mutant p53 protein. SAS cells were transfected with a vector carrying a mutant p53 gene (SAS/Trp248 cells) or neomycin resistant gene control vector (SAS/neo cells). Sensitivities of the transfected cells to X-ray or CDDP were measured with colony formation assay. The incidence of apoptosis by X-ray or CDDP was analyzed with Hoechst staining or DNA ladder formation assay. The activation of caspase-3 was estimated as an indicator of apoptosis by the detection of fragmentation of caspase-3 or poly (ADP ribose) polymerase (PARP) with Western blot. SAS/Trp248 cells showed X-ray- and CDDP-resistance due to the dominant negative nature of mutant p53, compared with SAS/neo cells. The incidence of DNA ladders and apoptotic bodies increased markedly in SAS/neo cells after X-ray irradiation or CDDP treatment, but increased only slightly in SAS/Trp248 cells. Fragmentation of caspase-3 and PARP was observed in SAS/neo cells, but almost no such fragmentation was observed in SAS/Trp248 cells after X-ray irradiation or CDDP treatment. The present results strongly suggest that the X-ray- and CDDP-sensitivities of human squamous cell carcinomas are p53-dependent, and that the sensitivities are tightly correlated with the induction of apoptosis through caspase-3 activation. The p53-dependent X-ray- or CDDP-sensitivity was supported by results from p53-null human lung cancer H1299 cells which were transfected with wild-type or mutant p53 gene.

Prevalence of Human Papillomavirus in Oropharyngeal Cancer: A Multicenter Study in Japan

Background: The incidence rates of oropharyngeal squamous cell carcinoma (OPSCC) have risen steadily in the USA and in northern Europe. These increases are thought to be a consequence of persistent infection with high-risk human papillomavirus (HPV) in OPSCC patients. HPV is an emerging etiologic factor in OPSCC. In Japan, the incidence of OPSCC has significantly increased over the last three decades. However, the population of HPV-positive OPSCC patients is currently unknown. We examined the nationwide trends with regard to HPV incidence in OPSCC patients at 21 specific sites, and examined the relationship between the presence of HPV and survival in OPSCC patients in Japan. Methods: Tumor samples were obtained from patients with OPSCC prior to treatment, and HPV infection was investigated by polymerase chain reaction (PCR). Hybrid Capture 2 (HC2) was also adopted for swab examination on the surface of fresh tumors. Results: HPV was detected by PCR in 79 (50.3%) out of 157 OPSCC patients. The clinical features of HPV-positive OPSCC were low differentiation, a tendency to involve the lateral wall, and high nodal staging. The sensitivity and specificity of HC2 were 93.7 and 96.2%, respectively, indicating its utility as a screening test. HPV-positive patients had significantly better overall survival and disease-free survival than HPV-negative patients.

DNA hypermethylation status of multiple genes in papillary thyroid carcinomas.

Objective: The aim of this study was hypermethylation of multiple genes for papillary thyroid carcinomas (PTCs). Methods: We examined 39 lesions using methylation-specific PCR to assess hypermethylation in genes, including p16^INK4a, p14^ARF, RB1, p27^Kip1and 0⁶-MGMT. Homozygous deletions of p16^INK4a and p14^ARF were investigated by differential PCR, all with reference to clinicopathological factors. Results: We foundmethylation of p16^INK4a in 35.9% (14/39); p14^ARF in 2.6% (1/39); RB1 in 23.1% (9/39); p27^Kip1 in 15.4% (6/39),and 0⁶-MGMT in 15.4% (6/39). Hypermethylation of at least one of these genes was apparent in 66.7% (26/39). Homozygous deletions of p14^ARF and p16^INK4a were detected in 7.7 (3/39) and 2.6% (1/39), respectively. In cases with p16^INK4a alterations, tumors were significantly increased. A history of chronic thyroid disease and lymphocytic infiltration was significantly associated with p14^ARF alterations, without regional lymph node metastases. Conclusions: Our data suggest that alterations in p16^INK4a, mainly hypermethylation, may be linked to tumor growth but not tumor development, while alterations in p14^ARF may contribute to the induction of chronic inflammation-related PTCs.

Glycerol as a chemical chaperone enhances radiation-induced apoptosis in anaplastic thyroid carcinoma cells

IntroductionAnaplastic thyroid carcinoma, which is one of the most aggressive, malignant tumors in humans, results in an extremely poor prognosis despite chemotherapy and radiotherapy. The present study was designed to evaluate therapeutic effects of radiation by glycerol on p53-mutant anaplastic thyroid carcinoma cells (8305c cells). To examine the effectiveness of glycerol in radiation induced lethality for anaplastic thyroid carcinoma 8305c cells, we performed colony formation assay and apoptosis analysis.ResultsApoptosis was analyzed with Hoechst 33342 staining and DNA ladder formation assay. 8305c cells became radiosensitive when glycerol was added to culture medium before X-ray irradiation. Apoptosis was induced by X-rays in the presence of glycerol. However, there was little apoptosis induced by X-ray irradiation or glycerol alone. The binding activity of whole cell extracts to bax promoter region was induced by X-rays in the presence of glycerol but not by X-rays alone.ConclusionThese findings suggest that glycerol is effective against radiotherapy of p53-mutant thyroid carcinomas.

Lack of p16/CDKN2 alterations in thyroid carcinomas

Exons 1-3 of the p16/CDKN2 gene and exons 4-9 of the p53 gene were screened for mutations by single-strand conformation polymorphism (SSCP) analysis and direct sequencing of PCR-amplified DNA from human primary thyroid carcinomas and thyroid carcinoma cell lines. The samples included 21 papillary carcinomas, 2 undifferentiated carcinomas, 1 follicular carcinoma, 1 medullary carcinoma and 2 cell lines originating from thyroid undifferentiated carcinomas. No homozygous deletions and mutations in the p16/CDKN2 were observed in any of the primary tumors or cell lined. In contrast, one of the two undifferentiated carcinomas an both cell lines demonstrated point mutations in the p53 gene. These results that p16/CDKN2 gene alteration is not required for malignant transformation in the thyroid, while p53 gene mutations may play a role in the progression from differentiated to undifferentiated carcinoma.

Different ras gene mutational frequencies in thyroid papillary carcinomas in Japan and Thailand

The incidence and pattern of ras oncogene mutations in human malignancies demonstrate geographic and racial differences. For example, specificity of alterations is found in cholangiocellular carcinomas in Thai patients with a different etiology from those in Japanese patients. In the present study, a comparison of ras gene mutations in thyroid papillary carcinomas from Japanese and Thai patients was performed using single-strand conformation polymorphism and direct sequencing analyses. The incidence of ras mutation differed markedly in Japanese (two of 24 carcinomas, 8.3%) and Thai (five of 10 carcinomas, 50%) patients. In addition, all but one ras mutation occurred at codon 12 of the K-ras gene in the Thai cases. These results suggest that thyroid cancers in Thailand may be due to specific genetic and/or environmental factors.

Snail-induced EMT promotes cancer stem cell-like properties in head and neck cancer cells.

Epithelial-mesenchymal transition (EMT) is a key process involved in the invasion and metastasis of cancer cells. Furthermore, EMT can induce a cancer stem cell (CSC)-like phenotype in a number of tumor types. We demonstrated that Snail is one of the master regulators that promotes EMT and mediates cancer cell migration and invasion in many types of malignancies including head and neck squamous cell carcinoma (HNSCC). In the present study, we investigated the role of Snail in inducing and maintaining CSC-like properties through EMT in HNSCC. We established HNSCC cell lines transfected with Snail. Stem cell markers were evaluated with real-time RT-PCR and western blot analysis. CSC properties were assessed using sphere formation and WST-8 assays as well as chemosensitivity and chick chorioallantoic membrane in vivo invasion assays. Introduction of Snail induced EMT properties in HNSCC cells. Moreover, Snail-induced EMT maintained the CSC-like phenotype, and enhanced sphere formation capability, chemoresistance and invasive ability. These data suggest that Snail could be one of the critical molecular targets for the development of therapeutic strategies for HNSCC.