Investigation of PTEN promoter methylation in ameloblastoma

Phosphatase and tensin homolog (PTEN) acts as a tumor suppressor gene. Inactivation of PTEN has been reported in various types of cancers. PTEN promoter methylation possibly underlies PTEN inactivation, which results in tumorigenesis. The aim of this study was to investigate whether PTEN promoter methylation contributes to PTEN inactivation in ameloblastoma and its associated protein expression. In total, 20 fresh-frozen ameloblastoma samples were evaluated for PTEN promoter methylation using methylation-specific polymerase chain reaction (MS-PCR). A subset of 10 paraffin-embedded ameloblastoma samples was examined for PTEN expression through immunohistochemistry. Four primary cultured ameloblastoma cells were investigated for PTEN promoter methylation and PTEN transcriptional expression via reverse transcription PCR. PTEN promoter methylation was detected in 65% (13/20) of the ameloblastoma samples. Of 10 ameloblastoma samples, 4 exhibited reduced PTEN expression. Of 5 samples with methylated PTEN, 3 (60%) were associated with loss of PTEN expression. However, PTEN expression was detected in 4 (80%) of 5 samples with unmethylated PTEN. In addition, 3 (75%) of 4 primary ameloblastoma cell cultures exhibited an inverse correlation between PTEN promoter methylation and PTEN transcription level. PTEN promoter methylation is found in a number of ameloblastomas but not significantly correlated with loss of PTEN expression. Genetic or epigenetic mechanisms other than PTEN promoter methylation may contribute to PTEN inactivation in ameloblastoma tumor cells

Odontogenic gene expression profile of human dental pulp-derived cells under high glucose influence: a microarray analysis

Hyperglycemia, a major characteristic of diabetes, is considered to play a vital role in diabetic complications. High glucose levels have been found to inhibit the mineralization of dental pulp cells. However, gene expression associated with this phenomenon has not yet been reported. This is important for future dental therapeutic application. Objective: Our study aimed to investigate the effect of high glucose levels on mineralization of human dental pulp-derived cells (hDPCs) and identify the genes involved. Methodology: hDPCs were cultured in mineralizing medium containing 25 or 5.5 mM D-glucose. On days 1 and 14, RNA was extracted and expression microarray performed. Then, differentially expressed genes (DEGs) were selected for further validation using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. Cells were fixed and stained with alizarin red on day 21 to detect the formation of mineralized nodules, which was further quantified by acetic acid extraction. Results: Comparisons between high-glucose and low-glucose conditions showed that on day 1, there were 72 significantly up-regulated and 75 down-regulated genes in the high-glucose condition. Moreover, 115 significantly up- and 292 down-regulated genes were identified in the high-glucose condition on day 14. DEGs were enriched in different GO terms and pathways, such as biological and cellular processes, metabolic pathways, cytokine–cytokine receptor interaction and AGE-RAGE signaling pathways. RT-qPCR results confirmed the significant expression of pyruvate dehydrogenase kinase 3 (PDK3), cyclin-dependent kinase 8 (CDK8), activating transcription factor 3 (ATF3), fibulin-7 (Fbln-7), hyaluronan synthase 1 (HAS1), interleukin 4 receptor (IL-4R) and apolipoprotein C1 (ApoC1). Conclusions: The high-glucose condition significantly inhibited the mineralization of hDPCs. DEGs were identified, and interestingly, HAS1 and Fbln-7 genes may be involved in the glucose inhibitory effect on hDPC mineralization

Evaluation of lymphocyte apoptosis in patients with oral cancer

Objectives: To evaluate apoptotic levels of peripheral blood mononuclear cells (PBMCs) and apoptotic regulatory proteins (Bax and Bcl-2) in lymphocyte subsets of oral cancer (OC) patients and healthy controls (HC). Methodology: The percentage of apoptotic cells and lymphocyte counts were measured in the first cohort using PBMCs obtained from 23 OC patients and 6 HC. In the second cohort, (OC, 33; HC, 13), the mean fluorescence intensity (MFI) of Bax and Bcl-2 in CD19+ B, CD4+ T, CD8+ T, and CD16+56+ natural killer (NK) cells was determined via flow cytometry. Results: The percentage of apoptotic cells was higher in the PBMCs of OC patients than in HC patients, particularly in patients with stage IV cancer (p<0.05). However, lymphocyte counts were significantly lower in stage IV patients (p<0.05). NK CD19+ B and CD16+56+ cell counts were significantly lower in OC patients compared with HC patients (p<0.001 and p<0.01, respectively), but CD4+ T cells were interestingly significantly higher in OC patients (p<0.001). While Bax MFI was slightly higher, Bcl-2 MFI was significantly lower for all four lymphocyte subsets in OC samples, particularly in stage IV patients, when compared with HC. Consequently, Bax/Bcl-2 ratios showed an upward trend from HC to OC patients, particularly those in stage IV. We found similar trends in Bax and Bcl-2 MFI for tumor stage, tumor size, and lymph node involvement. Conclusions: The increased lymphocyte apoptosis in stage IV OC patients may be related to higher Bax levels and lower Bcl-2 levels. The Bax/Bcl-2 ratio in lymphocytes may be useful to determine the prognosis of OC patients, and could be considered a mean for supportive treatment in the future

Cyclin A1 promoter hypermethylation in human papillomavirus-associated cervical cancer

BACKGROUND: The aim of this study was to evaluate epigenetic status of cyclin A1 in human papillomavirus-associated cervical cancer. Y. Tokumaru et al., Cancer Res 64, 5982-7 (Sep 1, 2004)demonstrated in head and neck squamous-cell cancer an inverse correlation between cyclin A1 promoter hypermethylation and TP53 mutation. Human papillomavirus-associated cervical cancer, however, is deprived of TP53 function by a different mechanism. Therefore, it was of interest to investigate the epigenetic alterations during multistep cervical cancer development. METHODS: In this study, we performed duplex methylation-specific PCR and reverse transcriptase PCR on several cervical cancer cell lines and microdissected cervical cancers. Furthermore, the incidence of cyclin A1 methylation was studied in 43 samples of white blood cells, 25 normal cervices, and 24, 5 and 30 human papillomavirus-associated premalignant, microinvasive and invasive cervical lesions, respectively. RESULTS: We demonstrated cyclin A1 methylation to be commonly found in cervical cancer, both in vitro and in vivo, with its physiological role being to decrease gene expression. More important, this study demonstrated that not only is cyclin A1 promoter hypermethylation strikingly common in cervical cancer, but is also specific to the invasive phenotype in comparison with other histopathological stages during multistep carcinogenesis. None of the normal cells and low-grade squamous intraepithelial lesions exhibited methylation. In contrast, 36.6%, 60% and 93.3% of high-grade squamous intraepithelial lesions, microinvasive and invasive cancers, respectively, showed methylation. CONCLUSION: This methylation study indicated that cyclin A1 is a potential tumor marker for early diagnosis of invasive cervical cancer

Antibiotic-Resistant Bacteria in Green Turtle (Chelonia mydas) Rearing Seawater

Antibiotic resistance of microorganisms is a serious health problem for both humans and animals. Infection of these bacteria may result in therapy failure, leading to high mortality rates. During an early intervention program process, the Sea Turtle Conservation Center of Thailand (STCCT) has faced high mortality rates due to bacterial infection. Previously, investigation of juvenile turtle carcasses found etiological agents in tissue lesions. Further determination of sea water in the turtle holding tanks revealed a prevalence of these causative agents in water samples, implying association of bacterial isolates in rearing water and infection in captive turtles. In this study, we examined the antibiotic resistance of bacteria in seawater from the turtle holding tank for a management plan of juvenile turtles with bacterial infection. The examination was carried out in three periods: 2015 to 2016, 2018, and 2019. The highest isolate numbers were resistant to beta-lactam, whilst low aminoglycoside resistance rates were observed. No gentamicin-resistant isolate was detected. Seventy-nine isolates (71.17%) were resistant to at least one antibiotic. Consideration of resistant bacterial and antibiotic numbers over three sampling periods indicated increased risk of antibiotic-resistant bacteria to sea turtle health. Essentially, this study emphasizes the importance of antibiotic-resistant bacterial assessment in rearing seawater for sea turtle husbandry

Human papillomavirus-related multiphenotypic sinonasal carcinoma: First case report associated with an intermediate-risk HPV type and literatures review

HPV-related Multiphenotypic sinonasal carcinoma (previously known as HPV-related carcinoma with adenoid cystic carcinoma-like features) is very rare tumor of which only 55 cases are reported. All cases to date are related to high-risk types of HPV including 16, 31, 33, 35 and 56. We report of a case of a nasal mass in a 50-year-old female. Biopsy demonstrated typical features of an HPV-related multiphenotypic sinonasal carcinoma, with both basaloid and ductal type cells. Tumor cells were arranged in lobulated sheets, with focal cribriform and microcystic growth patterns. The majority of tumor cells expressed myoepithelial markers (S-100 protein, smooth muscle actin and p63) and the ductal cells were immunopositive for CD117. Polymerase chain reaction and sequencing identified HPV type 26. HPV 26 becomes the first intermediate risk type that may be associated with HPV-related multiphenotypic sinonasal carcinoma. Keywords: HPV, Type 26, Sinonasal carcinoma, Multiphenotypi

Efficiency of CIN2+ Detection by Thyrotropin-Releasing Hormone (TRH) Site-Specific Methylation

Cervical cancer screening typically involves a Pap smear combined with high-risk human papillomavirus (hr-HPV) detection. Women with hr-HPV positivity but normal cytology, as well as those with precancerous abnormal cytology, such as low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL), are referred for colposcopy and histology examination to identify abnormal lesions, such as cervical intraepithelial neoplasia (CIN) and cervical cancer. However, in order to enhance the accuracy of detection, bioinformatics analysis of a microarray database was performed, which identified cg01009664, a methylation marker of the thyrotropin-releasing hormone (TRH). Consequently, a real-time PCR assay was developed to distinguish CIN2+ (CIN2, CIN3, and cervical cancer) from CIN2- (CIN1 and normal cervical epithelia). The real-time PCR assay utilized specific primers targeting methylated cg01009664 sites, whereas an unmethylated reaction was used to check the DNA quality. A cut-off value for the methylated reaction of Ct < 33 was established, resulting in improved precision in identifying CIN2+. In the first cohort group, the assay demonstrated a sensitivity of 93.7% and a specificity of 98.6%. In the cytology samples identified as atypical squamous cells of undetermined significance (ASC-US) and LSIL, the sensitivity and specificity for detecting CIN2+ were 95.0% and 98.9%, respectively. However, when self-collected samples from women with confirmed histology were tested, the sensitivity for CIN2+ detection dropped to 49.15%, while maintaining a specificity of 100%. Notably, the use of clinician-collected samples increased the sensitivity of TRH methylation testing. TRH methylation analysis can effectively identify women who require referral for colposcopy examinations, aiding in the detection of CIN2+

Alteration of Extracellular Matrix Components in the Anterior Pituitary Gland of Neonatal Rats Induced by a Maternal Bisphenol A Diet during Pregnancy

The extracellular matrix (ECM) plays crucial roles in the anterior pituitary gland via the mechanism of cell–ECM interaction. Since bisphenol A (BPA), a well-known endocrine disruptor, can cross through the placenta from mother to fetus and bind with estrogen receptors, cell populations in the neonatal anterior pituitary gland could be the target cells affected by this chemical. The present study treated maternal rats with 5000 µg/kg body weight of BPA daily throughout the pregnancy period and then investigated the changes in ECM-producing cells, i.e., pericytes and folliculostellate (FS) cells, including their ECM production in the neonatal anterior pituitary at Day 1. We found that pericytes and their collagen synthesis reduced, consistent with the increase in the number of FS cells that expressed several ECM regulators—matrix metalloproteinase (MMP) 9 and the tissue inhibitors of metalloproteinase (TIMP) family. The relative MMP9/TIMP1 ratio was extremely high, indicating that the control of ECM homeostasis was unbalanced. Moreover, transmission electron microscopy showed the unorganized cell cluster in the BPA-treated group. This study revealed that although the mother received BPA at the “no observed adverse effect” level, alterations in ECM-producing cells as well as collagen and the related ECM balancing genes occurred in the neonatal anterior pituitary gland

Patterns and Possible Roles of LINE-1 Methylation Changes in Smoke-Exposed Epithelia

<div><p>Tobacco smoking and reduced methylation of long interspersed element-1 (LINE-1) are crucial in oral carcinogenesis. 5′UTR of human LINE-1 sequence contains several CpG dinucleotides which are methylated in various proportions (0–100%). Methylation levels of many LINE-1s in cancer were reduced, hypomethylated. The hypomethylation of each LINE-1 locus can promote instability of genome and repress expression of a gene located on that same chromosome. This study investigated if cigarette smoking influences LINE-1 methylation of oral mucosal cells. The methylation of human LINE-1 in clinically normal oral mucosa of current smokers was compared to non-smokers. By using the combined bisulphite restriction analysis, each LINE-1 sequence was categorised into 4 patterns depending on the methylation status and location of the two 18-bp successive CpG from 5′ to 3′ including ^mC^mC, ^uC^uC, ^mC^uC and ^uC^mC. Of these, ^mC and ^uC represent methylated and unmethylated CpG, respectively. The DNA bisulphite sequence demonstrated that most CpGs of ^mC^mC and ^uC^uC were methylated and unmethylated, respectively. Nevertheless, some CpGs of each ^mC^uC or ^uC^mC allele were methylated. Imaging of the digestion products was used to generate %methylation value. No significant difference in the overall LINE-1 methylation level but the differences in percentages of some methylation patterns were discovered. The %^mC^mC and %^uC^uC increased, while the %^mC^uC decreased in current smokers (<em>p</em> = 0.002, 0.015, and <0.0001, respectively). Additionally, the lower %^mC^uC still persisted in persons who had stopped smoking for over 1 year (<em>p</em> = 0.001). The %^mC^uC also decreased in the higher pack-year smokers (<em>p</em> = 0.028). Smoking possibly altered ^mC^uC to ^mC^mC and ^uC^uC forms, and changes ^uC^mC to ^uC^uC forms. In conclusion, smoking changes methylation levels of partial methylated LINE-1s and increased the number of hypo- and hypermethylated loci. These hypomethylated LINE-1s may possess carcinogenesis potential. Moreover, LINE-1 methylation patterns may be useful for monitoring oral carcinogenesis in smokers.</p> </div