Cell kinetic study of the endometrium by nonisotopic in situ hybridization for histone H3 messenger RNA and immunohistochemistry for Ki‐67 and for estrogen and progesterone receptors

We investigated the cell kinetics of the endometrium in hysterectomy specimens taken for leiomyoma from 22 women with regular ovulatory menstrual cycles. Formalin‐fixed, paraffin‐embedded tissue sections were examined for proliferating activity using histone H3 messenger RNA in situ hybridization (H3 mRNA‐ISH) and immunostaining for the Ki‐67 antigen. The relationship of the proliferative activity of endometrial cells to the immunohistochemical expression of the estrogen receptor (ER) and the progesterone receptor (PR) was also examined. During the menstrual cycle, H3 mRNA expression was observed in both the epithelial cells and the stromal cells of the endometrium. In the functional layer, the labeling indices for H3 mRNA (H3 mRNA‐LIs) in the epithelial cells peaked in the late proliferative phase, decreased sharply in the early secretory phase, and remained unchanged thereafter. On the other hand, H3 mRNA‐LIs of stromal cells displayed two peaks: one in the midproliferative phase and the other in the late secretory phase, the former peak being the greater. In the basal layer, epithelial cells and stromal cells showed low H3 mRNA‐LIs and no significant variation throughout the menstrual cycle. The H3 mRNA‐LIs correlated well with the Ki‐67‐LIs and were lower than the corresponding Ki‐67‐LIs. The regression coefficient (H3 mRNA‐LIs against the Ki‐67‐LIs) was 0.33 for epithelial cells and 0.49 for stromal cells, suggesting that the cell cycle time was longer for epithelial cells than for stromal cells. The proliferative activity of endometrial cells showed close relationships with the expressions of ER and PR in the endometrium. When used in combination with other proliferative markers in paraffin‐embedded tissue sections, H3 mRNA‐ISH could open broader perspectives on the cell kinetics of the endometrium. Anat Rec 266:234–240, 2002. © 2002 Wiley‐Liss, Inc.     

Human telomerase catalytic subunit gene re-expression is an early event in oral carcinogenesis

AIMS: Detection of telomerase catalytic subunit (hTERT) mRNA has been used as a surrogate marker for estimation of telomerase activity. The exact role and timing of telomerase re-activation, a key enzyme implicated in cellular immortalization and transformation, in the multistep process of oral carcinogenesis is still unknown. The aim was to test the hypothesis that (i) quantitative rather than qualitative differences exist in the level of hTERT mRNA expression between normal oral mucosa, different grades of oral epithelial abnormalities and squamous cell carcinomas of the oral cavity, and that (ii) hTERT gene re-expression is an important, probably early event in oral carcinogenesis. METHODS AND RESULTS: The relative quantity of hTERT mRNA was analysed in 45 frozen oral epithelia representing different morphological stages of oral carcinogenesis classified according to the Ljubljana classification and in 37 oral squamous cell carcinomas, using a commercially available LightCycler Telo TAGGG hTERT Quantification kit. hTERT mRNA was not detected in normal or reactive hyperplastic oral epithelia, but was present in 43% of atypical hyperplasias (premalignant lesions), 60% of intraepithelial carcinomas and 68% of oral squamous cell carcinomas. Statistical analysis revealed two groups of oral epithelial changes, with significant differences in the levels of hTERT mRNA expression: 1, normal and reactive hyperplastic oral epithelium, and 2, atypical hyperplasia, intraepithelial carcinomas and squamous cell carcinomas. CONCLUSION: These data suggest that hTERT gene re-expression represents an early event in the multistep process of oral carcinogenesis, already detectable at the stage of precancerous oral epithelial changes. Nevertheless, other genetic aberrations appear to be necessary for progression of oral epithelial abnormalities towards invasive squamous cell carcinoma.     

Protective Effect of Withaferin-A on Micronucleus Frequency and Detoxication Agents During Experimental Oral Carcinogenesis

Our aim was to investigate the effect of Withaferin-A on bone marrow micronucleus frequency and buccal mucosa detoxication agents during 7, 12-dimethylbenz[a]anthracene (DMBA) induced hamster buccal pouch carcinogenesis. Oral squamous cell carcinoma was developed in hamsters'' buccal pouches by painting 0.5% DMBA in liquid paraffin, three times per week for 14 weeks. We observed 100% tumor formation in DMBA painted hamsters. Elevated frequency of bone marrow micronucleated polychromatic erythrocytes (MnPCEs) and decrease in buccal mucosa phase II detoxication agents were noticed in tumor bearing hamsters. Oral administration of Withaferin-A significantly reduced the micronucleus frequency and brought back the status of phase II detoxication agents in DMBA painted hamsters. Our study thus demonstrated the protective effect of Withaferin-A on DMBA-induced micronucleus frequency in the bone marrow of golden Syrian hamsters. Also, Withaferin-A maintained the status of buccal mucosa detoxication agents during DMBA-induced hamster buccal pouch carcinogenesis.     

Thymosin ß4 expression in colorectal polyps and adenomas

OBJECTIVE:

Thymosin beta 4 (Tβ4) is a ubiquitous peptide that plays pivotal roles in the cytoskeletal system and in cell differentiation. Recently, a role for Tβ4 has been proposed in experimental and human carcinogenesis, including gastrointestinal cancer. This study was aimed at evaluating the relationship between Tβ4 immunoreactivity and the initial steps of carcinogenesis.

METHODS:

In total, 60 intestinal biopsies, including 10 hyperplastic polyps, 10 sessile serrated adenomas/polyps, 15 colorectal adenomas with low-grade dysplasia, 15 adenomas with high-grade dysplasia, 15 adenocarcinomas and 10 samples of normal colon mucosa, were analyzed for Tβ4 expression by immunohistochemistry.

RESULTS:

Weak cytoplasmic reactivity for Tβ4 was detected in the normal colon mucosa. No reactivity for Tβ4 was found in hyperplastic and sessile serrated polyps/adenomas. Tβ4 expression was observed in 10/15 colorectal adenocarcinomas. In adenomas with low-grade dysplasia, Tβ4 immunoreactivity was mainly detected in dysplastic glands but was absent in hyperplastic glands. Tβ4 immunoreactivity was characterized by spot-like perinuclear staining. In high-grade dysplastic polyps, immunostaining for Tβ4 appeared diffuse throughout the entire cytoplasm of dysplastic cells. Spot-like perinuclear reactivity was detected in adenocarcinoma tumor cells.

CONCLUSIONS:

Our study shows for the first time that Tβ4 is expressed during different steps of colon carcinogenesis. The shift of Tβ4 immunolocalization from low-grade to high-grade dysplastic glands suggests a role for Tβ4 in colorectal carcinogenesis. However, the real meaning of Tβ4 reactivity in dysplastic intestinal epithelium remains unknown.     

Histone gene (H3) expression in chemically transformed oral keratinocytes

The hamster cheek pouch is an excellent target tissue for the experimental study of oral carcinogenesis. In the course of searching for molecular alterations during the malignant transformation process, the necessity for a molecular marker for cellular proliferation became apparent. In this report, we show that the cellular level of the histone H3 mRNA is valid as a molecular index of proliferation for cycling cell populations. H3 is known to be proliferation dependent for its expression in cultured animal cells. This study shows that H3 retains its cell-cycle-dependent expression in chemically transformed oral keratinocytes. The onset of H3 mRNA synthesis couples to the onset of DNA synthesis (S-phase). The cellular level of H3 mRNA therefore is proportional to the fraction of cells in the S-phase of the cell cycle. This conveniently allows us to correlate, in asynchronized cell populations, the expression of cellular genes to their proliferation rates. We demonstrate the usefulness of this proliferation marker by presenting data that different chemically induced oral carcinomas, but not normal cheek pouch tissues, contain readily detectable levels of c-Ki-ras proto-oncogene mRNA. Probing the same RNA blot to quantitate H3 mRNA levels allowed us to conclude that the high levels of c-Ki-ras mRNA in tumor tissues was likely due to the increased growth rate of the tumor tissues and not due to the deregulated expression of this cellular-proto-oncogene.     

Overexpression of phosphorylated histone H3 is an indicator of poor prognosis in gastric adenocarcinoma patients.

Ki-67 immunostaining is commonly used for assessing cell proliferation, but studies of its use as a prognostic indicator have revealed discordant results in gastric cancer patients. Recently, antibodies for phosphorylated histone H3 have been used to identify dividing cells because of its precise overexpression in mitosis. The authors tested the hypothesis that phosphorylated histone H3 overexpression might be a good prognostic indicator for gastric cancer patients by conducting an immunohistochemical comparison with Ki-67 in gastric cancer samples. One hundred twenty-two surgically resected primary cases were selected and histologically categorized in accordance with Lauren's classification. No correlation was found between phosphorylated histone H3 and Ki-67 regarding overexpression. However, correlations between phosphorylated histone H3 overexpression and clinicopathologic variables were noted for histologic type (intestinal type predominant in high labeling indices [LIs], defined as over the value of the 75th percentile; P<0.01), vessel invasion (positive in high LIs; P=0.05), and lymph node metastasis (positive in high LIs; P=0.04). With regard to Ki-67 overexpression, no correlation was evident with the clinicopathologic variables except histologic type (intestinal type predominant; P=0.05). By the Kaplan-Meier method with the log-rank test, cases overexpressing phosphorylated histone H3 showed a poorer prognosis than cases with low expression (P<0.01). In contrast, Ki-67 expression did not influence prognosis. Multivariate analyses indicated phosphorylated histone H3 overexpression to be an independent prognostic factor, together with lymphatic invasion and venous invasion (P<0.01). In conclusion, it seems likely that phosphorylated histone H3 plays an important role in the prognosis of gastric cancer, and its immunohistochemical investigation is useful for the prediction of prognosis in gastric cancer.     

Ber-EP4 immunoreactivity depends on the germ layer origin and maturity of the squamous epithelium

AIM: To map the expression of Ber-EP4 in well-differentiated squamous epithelia, metaplastic squamous epithelia and dysplastic squamous epithelia of different origins. METHODS AND RESULTS: Squamous epithelium of different origin was stained using a standard immunohistochemistry method applied to paraffin sections. We found that normal squamous epithelium of the oral cavity, oesophagus, uterine cervix, vagina, anal canal, and branchial cysts are Ber-Ep4-negative, as are the mature squamous metaplasia of bronchial mucosa, urinary bladder mucosa and uterine cervical mucosa. In contrast, immature squamous metaplasia of bronchial mucosa, or uterine cervical mucosa, and squamous dysplasia of oral mucosa of endodermal origin, or uterine cervical mucosa in most cases expressed Ber-EP4. CONCLUSION: Squamous epithelia of ectodermal origin never express Ber-EP4, whether normal, hyperplastic, dysplastic or neoplastic. In contrast, squamous epithelium of endodermal origin sometimes contains the target glycoproteins of Ber-EP4 when immature, metaplastic, dysplastic or neoplastic. The results indicate that the differences in expression of Ber-EP4 in squamous epithelium depend primarily on germ layer origin, and on the maturity of the epithelium.     

Telomerase reactivation is an early event in laryngeal carcinogenesis.

The exact role and timing of reactivation of telomerase, a key enzyme implicated in cellular immortalization and transformation in the multistep process of laryngeal carcinogenesis, is still unknown. We attempted to (1) determine that quantitative differences exist in the levels of telomerase catalytic subunit (hTERT) mRNA expression among different grades of laryngeal epithelial abnormalities classified according to the Ljubljana classification; (2) determine that telomerase reactivation is an important, most probably early event in laryngeal carcinogenesis; and (3) analyze whether the relative quantity of hTERT mRNA can be used as a molecular biomarker in the early detection of precancerous lesions. The relative quantity of hTERT mRNA, expressed as an hTERT index, was analyzed in 140 frozen laryngeal tissue specimens representing different morphological stages of laryngeal carcinogenesis by using a commercially available LightCycler Telo TAGGG hTERT Quantification kit. The presence and relative quantity of hTERT mRNA in laryngeal epithelium increases progressively with the degree of epithelial abnormalities. hTERT mRNA was detectable in 1/15 normal laryngeal epithelia (7%, mean hTERT index 0.02), 3/15 simple hyperplasias (20%, mean hTERT index 0.09), 10/27 abnormal hyperplasias (37%, mean hTERT index 0.18), 9/12 atypical hyperplasias (75%, mean hTERT index 0.74), 8/9 intraepithelial carcinomas (89%, mean hTERT index 1.82), and 53/62 invasive laryngeal squamous cell carcinomas (85%, mean hTERT index 2.51). Statistical analysis revealed two groups of laryngeal epithelial changes with significant differences in the levels of hTERT mRNA expression (P <.0033): (1) normal and reactive hyperplastic laryngeal epithelium (simple and abnormal hyperplasia) and (2) atypical hyperplasia (precancerous lesion), intraepithelial and invasive laryngeal squamous cell carcinoma. The results of the present study suggest that telomerase reactivation is an early event in laryngeal carcinogenesis, detectable already at the stage of precancerous laryngeal epithelial changes. Nevertheless, other genetic abnormalities appear to be necessary for progression of these epithelial abnormalities toward invasive laryngeal squamous cell carcinoma.     

Protective effect of ferulic acid on 7,12-dimethylbenz[a]anthracene-induced skin carcinogenesis in Swiss albino mice

Our aim was to evaluate and compare the chemopreventive potential of topically applied and orally administered ferulic acid in 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin carcinogenesis. Estimating the status of phase I and phase II detoxication agents, lipid peroxidation byproducts and antioxidants during DMBA-induced skin carcinogenesis assessed the mechanistic pathway for its chemopreventive efficacy. Skin squamous cell carcinoma was induced in the shaved back of mice, by painting with DMBA (25 μg in 0.1 mL^?1 acetone) twice weekly for 8 weeks. We have observed 100% tumor formation in the 15th week of experimental period in mice treated with DMBA alone. Marked alterations in the status of phase I and phase II detoxication agents, lipid peroxidaton byproducts and antioxidants were observed in tumor bearing mice. Oral administration of ferulic acid completely prevented the formation of skin tumors, whereas topically applied ferulic acid did not show significant chemopreventive activity during DMBA-induced mouse skin carcinogenesis. Also, oral administration of ferulic acid reverted the status of phase I and phase II detoxication agents, lipid peroxidaton byproducts and antioxidants to near-normal range in DMBA-treated mice. Our results thus demonstrate that orally administered ferulic acid has potent suppressing effect on cell proliferation during DMBA-induced skin carcinogenesis. This is probably due to its modulating effect on the status of lipid peroxidation, antioxidants and detoxication agents during DMBA-induced skin carcinogenesis.     

二甲基苯并蒽涂抹联合创伤诱发舌癌模型的比较研究

目的为探讨人舌癌的发生和发展规律,建立与人相近的鼠舌癌模型,并进行 SD 大鼠与金黄地鼠致癌表现差异的研究。方法 1%二甲基苯并蒽(DMBA)丙酮液涂抹联合应用舌创伤致 SD 大鼠和金黄地鼠左侧舌癌,共 24 周,通过光镜和透射电镜动态观察舌病理学改变。结果 SD 大鼠和金黄地鼠 3 d 后舌背黏膜左侧缘相继出现溃疡、白斑、肿大。SD大鼠 8 周可见上皮层增生和基底膜细胞的异型性改变,16 周癌变的基底细胞突破基底膜向深层浸润, 镜下见癌细胞分化程度差,无角化珠形成,致癌率 76.6%;金黄地鼠 8 周出现黏膜白斑或乳头状突起,继之出现黏膜糜烂,10 周逐渐形成外生性肿块,镜下见癌细胞分化程度高,有角化珠形成,致癌率为 93.3%。结论 DMBA 涂抹联合创伤法均可在 SD 大鼠和金黄地鼠诱发舌癌,成功率高,适合用于舌癌的相关研究。金黄地鼠比 SD 大鼠致癌率高,金黄地鼠主要表现为高分化癌,SD 大鼠则表现为低分化癌。     

A quantitative study of lamina densa alterations in hamster cheek pouch carcinogenesis

Lesions produced by topical application of 0.5 per cent. DMBA to the hamster cheek pouch epithelium were classified as hyperplasia, dysplasia and carcinoma groups using strict histological criteria. Untreated epithelium served as a control. Tissue samples from five animals in each group were processed for electron microscopy and electron micrographs from the epithelialconnective tissue junction were obtained from 5 blocks per animal. The micrographs were subjected to stereological intersection counting to determine the relative surface (S_SLD,BM) of lamina densa which was in normal relationship to the basal cell plasma membranes. Quantitative results indicated a progressive loss of lamina densa during carcinogenesis and this was accompanied by the extrusion of pseudopodia from the basal cells through the gaps. The pseudopodia were frequently related to peripheral cytoplasmic microfilaments. Quantitative data confirmed the progressive nature of this loss, with values for S_SLD,BM being of the order of 98 per cent., 88 per cent., 76 per cernt. and 42 per cent. for normal epithelium and for the hyperplastic, dysplastic and carcinomatous lesions respectively. The loss of lamina densa is discussed in relation to the specificity of the response and to the development of features indicative of motility in transforming cells.     

Expression of the 3-fucosyl N-acetyllactosamine (CD 15) antigen in normal, metaplastic, dysplastic, and neoplastic squamous epithelia

The 3-fucosyl N-acetyllactosamine residue is the antigen recognized by the monoclonal antibody MC2. Using MC2, we demonstrated the distribution of this antigen in a variety of squamous epithelia. The antigen is expressed to a variable degree on supra-basal cells in most normal non-keratinizing squamous mucosae, with a similar distribution in metaplastic squamous epithelia; antibody-labelled latex microspheres and immunogold electron microscopy show the antigen to form part of the glycocalyx. In dysplastic and neoplastic squamous lesions, expression is reduced or absent except in cells around areas of differentiation. Prior neuraminidase treatment of sections had little effect on the amount or distribution of demonstrable antigen. Expression of this antigen by cells in non-keratinizing squamous epithelia gives an indication of cell maturity and may provide a histological marker for the grading of dysplastic and malignant squamous mucosal lesions. A possible role for these carbohydrate residues in squamous mucosal defence is discussed.     

Induction of cell proliferation, micronuclei and hyperdiploidy/polyploidy in the mammary cells of DDT- and DMBA-treated pubertal rats

The environmental estrogen, dichlorodiphenyltrichloroethane (DDT), and its metabolites have been implicated in the development of breast cancer through mechanisms that remain to be elucidated. It has been hypothesized that exposure to DDT and its metabolites, during critical periods of development, can contribute to an elevated risk for breast cancer in adults. In the present study, we have investigated the effect of o,p'-DDT on mammary gland cell proliferation and chromosomal alterations, in a rat mammary cancer model (commonly used to study human cancer), to gain insights into its potential role in the development of breast cancer. Twenty-one-day-old female Sprague-Dawley (SD) rats were administered o,p'-DDT, 7,12-dimethylbenz[a]anthracene (DMBA), genistein, DDT+DMBA, or DDT+DMBA+genistein, over a 14-day period. To determine changes in chromosome number and structure, we used the micronucleus assay as well as multicolor fluorescence in situ hybridization (FISH) region-specific DNA probes for rat chromosomes 4 and 19. Cell proliferation was evaluated using 5-bromo-2'-deoxyuridine (BrdU). Significant increases in BrdU-incorporated cells were seen in the rats treated with DDT+DMBA. Although micronucleus frequencies were somewhat elevated in several of the treatment groups, significant increases were not seen in any of them. Significant increases in numerical chromosomal aberrations were detected in all of the DDT- and DMBA-treated groups. Genistein significantly reduced BrdU incorporation and polyploidy in the DDT+DMBA-treated rats. These initial studies indicate that DDT and DMBA can induce cellular and chromosomal alterations in the rat mammary gland, which is consistent with the hypothesis that these agents can induce early events in mammary carcinogenesis.     

Telomerase catalytic subunit in laryngeal carcinogenesis--an immunohistochemical study.

We recently demonstrated that (1) telomerase catalytic subunit messenger RNA (mRNA) relative quantities increase progressively with the degree of laryngeal epithelial abnormalities and that (2) telomerase catalytic subunit gene re-expression represents an early event in laryngeal carcinogenesis. The aim of the study was to determine whether telomerase catalytic protein immunohistochemisty reflects telomerase catalytic subunit gene expression in different grades of laryngeal epithelial abnormalities and squamous cell carcinomas of the larynx. Telomerase catalytic protein was analysed immunohistochemically in 106 laryngeal epithelial tissue samples: 10 normal epithelia, 15 squamous cell hyperplasias, 14 basal/parabasal cell hyperplasias, 10 atypical hyperplasias, eight intraepithelial carcinomas and 49 squamous cell carcinomas. At least 200 nuclei of each lesion were quantified per slide and the number of positive signals per nucleus was expressed as a telomerase catalytic protein index. The mean telomerase catalytic protein index increased progressively with the degree of laryngeal epithelial abnormalities: from 0.17 in normal epithelia, 0.44 in squamous cell hyperplasia, 0.54 in basal/parabasal cell hyperplasia, 0.91 in atypical hyperplasia, 1.05 in intraepithelial carcinoma to 0.96 in squamous cell carcinomas. Statistical analysis revealed three different groups of laryngeal epithelial changes according to the number of telomerase catalytic protein signals per nucleus: (1) normal epithelium, (2) regenerative epithelium (squamous cell hyperplasia, basal/parabasal cell hyperplasia), and (3) atypical hyperplasia, intraepithelial carcinoma and squamous cell carcinoma (P<0.0033). Telomerase catalytic protein immunohistochemistry parallels well with telomerase catalytic subunit mRNA relative quantities in laryngeal carcinogenesis. In normal and regenerative laryngeal epithelia, telomerase catalytic protein is present in occasional basal/parabasal nuclei, becomes undetectable with maturation or differentiation of epithelial cells, and reflects the regenerative capacity of squamous epithelium. Nevertheless, several telomerase catalytic protein signals in the majority of nuclei in precancerous lesions, intraepithelial carcinomas and squamous cell carcinomas, are consistent with telomerase catalytic subunit gene re-expression, an early event in laryngeal carcinogenesis.     

Overexpression of human telomerase RNA is an early event in oesophageal carcinogenesis

 Telomerase, the ribonucleoprotein enzyme that elongates telomeres, is repressed in normal human somatic cells but is reactivated during tumour progression. The purpose of this study was to investigate the localization of human telomerase RNA (hTR) expression in human oesophageal dysplasia and cancer by using in situ mRNA hybridization (ISH) with avidin–biotin staining. Ki-67 immunoreactivity was also examined. We analysed 51 squamous cell carcinomas, 9 dysplasias and 60 normal mucosae. The integrity of the mRNA in each sample was verified by using a poly d(T)₂₀ probe. Seventy-six samples (63%) showed no mRNA degradation; these included 30 carcinomas, 7 dysplasias and 39 normal mucosae. At the single-cell level, high levels of hTR expression were found in the cytoplasm and especially in the nucleus. Most (>90%) cancer cells demonstrated high levels of hTR expression in 29 (97%) of the 30 tumours. Most dysplastic cells also showed high levels of hTR in all 7 dysplastic cases. In all 39 normal mucosae, most basal cells indicated high levels of hTR expression, which were also seen in infiltrating lymphocytes. The distribution of hTR-expressing cells was similar to that of Ki-67-positive cells. These data suggest that overexpression of hTR may be correlated with the proliferative activity that defined by Ki-67 immunoreactivity and is an early event in carcinogenesis of the oesophagus. Received: 14 December 1998 / Accepted: 20 January 1999     

A comparative study of digoxigenin, 2,4-dinitrophenyl, and alkaline phosphatase as deoxyoligonucleotide labels in non-radioisotopic in situ hybridisation.

AIM: To determine the optimum form of labelling and the most efficient reporter molecule for non-radioisotopic in situ hybridisation (ISH). METHODS: Nine deoxyoligonucleotides complementary to histone mRNA were synthesised and labelled either enzymatically or during solid-phase synthesis with the reporter molecules digoxigenin, 2,4-dinitrophenyl (DNP), or alkaline phosphatase. Pooled deoxyoligonucleotide cocktails were then used in non-radioisotopic ISH detection of histone mRNA in human tonsil. Hybrid detection was by nitroblue tetrazoleum/5-bromo-4-chloro-3-indolyl phosphate colorimetric development. RESULTS: The use of a spacer in 3' enzymatic labelling and when labelling with alkaline phosphatase significantly increased ISH signal. The 3' and 5' labelling of oligonucleotides with triple DNP groups during solid-phase synthesis produced the strongest signal as determined by the highest cell signal intensity and shortest development time. CONCLUSIONS: 3' and 5' solid-phase labelling with triple DNP groups produced the best labelling for non-isotopic ISH using deoxyoligonucleotide cocktails.     

Patterns of global DNA and histone methylation appear to be similar in normal, dysplastic and neoplastic oral epithelium of humans

Although there is growing interest in the possibility that alterations in histone methylation may play a role in carcinogenesis, it has not been explored adequately in humans. Similarly, there are no reports of associations between this and a similar epigenetic event, DNA methylation. Using immunohistochemical staining, we compared the methylation of DNA and histones in histopathologically normal oral epithelium, dysplastic oral lesions, and squamous cell cancers (SCCs) from subjects with squamous cell cancer (n=48) with those of normal oral epithelium from subjects without oral cancer (n=93) who were matched on age and race. Monoclonal antibodies specific for 5 methyl cytosine (5-mc), lysine 4 of histone H_3 (H_3-Lys4), and lysine 9 of histone H_3 (H_3-Lys9) were used in this study. The percentages of cells positive and a weighted average of the immunostaining intensity scores were calculated for each of these tissues, and Spearman correlation analyses were employed to study associations between DNA and histone methylation. Correlations between DNA and histone methylation, H_3-Lys4 and H_3-Lys9 were positive and statistically significant in all tissue types; they were strongest in normal oral epithelium from non-cancer subjects (r=0.63, p < 0.001 and r=0.62, p < 0.001 respectively). Similarly, the positive correlations between H_3-Lys4 and H_3-Lys9 were statistically significant in all tissue types and strongest in normal oral epithelium from non-cancer subjects (r=0.77, p< 0.001). Patterns of DNA and histone methylation are similar in tissues across the spectrum of oral carcinogenesis, and there is a significant positive association between these two epigenetic mechanisms.     

穿心莲内酯对口腔癌生长的影响

 目的：探讨穿心莲内酯对口腔癌的作用及其机制。方法：用穿心莲内酯及其对照药物治疗金黄地鼠颊囊癌,通过测量肿瘤体积观察治疗效果。用免疫组化检测穿心莲内酯对肿瘤血管和肿瘤细胞增殖的作用,用TUNEL法检测穿心莲内酯对肿瘤细胞凋亡的作用,用免疫组化检测凋亡信号分子caspase-3的表达情况。结果：穿心莲内酯治疗组颊囊癌的肿瘤体积明显比对照组小,肿瘤微血管密度和增殖的肿瘤细胞也明显低于对照组,肿瘤细胞的凋亡率明显高于对照组,而且治疗组中caspase-3明显被活化。结论:穿心莲内酯通过抑制肿瘤血管生成和肿瘤细胞增殖以及促进肿瘤细胞凋亡,抑制了金黄地鼠颊囊癌的生长。穿心莲内酯通过caspase-3途径诱导了肿瘤细胞的凋亡。