Downregulation of genes encoding for subunits of adaptor complex-3 in cervical carcinomas

We explored the expression of four genes encoding for subunits of AP-3 in cervical tumors and cancer cell lines. Using RT-PCR we demonstrated more than twofold decrease in the levels of mRNA of AP3D1, AP3B1, AP3M1, and AP3S1 in 32, 28, 23, and 26% tumors in comparison with normal tissues of uterine cervix, respectively. The level of mRNA of at least one subunit was decreased in 28 out of 47 (60%) of tumors and in four out of five cancer cell lines in comparison to tissues adjacent to tumors. The suppression of expression of any of the subunits was revealed in 15 out of 28 cases (54%). The expression of two and more subunits was decreased simultaneously in different combinations in 13 cases (46%). This fact testifies to the lack of a common mechanism of downregulation of four subunits in tumors. There is a tendency to more frequent suppression of AP-3A expression in tumors associated with lymphatic node metastases as compared with tumors without metastases (P = 0.034). Thus, here we demonstrate for the first time the decrease in expression of genes encoding for AP-3A subunits in tumors.     

AEBP1在结直肠癌中的表达及其意义

目的:研究脂肪细胞增强结合蛋白1(AEBP1)在结直肠癌组织中的表达情况,以探索其临床意义。方法:运用免疫组织化学和实时定量PCR技术,对AEBP1在结直肠癌中的表达情况进行检测,并分析其表达量与患者临床病理特征的关系。结果:AEBP1在结直肠正常组织、腺瘤和癌组织中的免疫评分有明显差异(P0.05),而且AEBP1的阳性表达与肿瘤的分期和浸润深度明显相关。基因水平AEBP1的表达在肿瘤组织中明显高于正常组织。结论:AEBP1在结直肠癌组织中存在异常表达,并且可能是结直肠癌早期诊断和浸润深度的肿瘤标志物。     

Aberrant Expression of Osteopontin and E-Cadherin Indicates Radiation Resistance and Poor Prognosis for Patients with Cervical Carcinoma

Radiotherapy is the first-line treatment for all stages of cervical cancer, whether it is used for radical or palliative therapy. However, radioresistance of cervical cancer remains a major therapeutic problem. Consequently, we explored if E-cadherin (a marker of epithelial-mesenchymal transition) and osteopontin could predict radioresistance in patients with locally advanced cervical squamous cell carcinoma (LACSCC). Patients were retrospectively reviewed and 111 patients divided into two groups (radiation-resistant and radiation-sensitive groups) according to progression-free survival (PFS). In pretreated paraffin-embedded tissues, we evaluated E-cadherin and osteopontin expression using immunohistochemical staining. The percentage of patients with high osteopontin but low E-cadherin expression in the radiation-resistant group was significantly higher than those in the radiation-sensitive group (p<0.001). These patients also had a lower 5-year PFS rate (p<0.001). Our research suggests that high osteopontin but low E-cadherin expression can be considered as a negative, independent prognostic factor in patients with LACSCC ([Hazard ratios (95% CI) 6.766 (2.940, 15.572)], p<0.001).     

Potassium Currents in Freshly Dissociated Uterine Myocytes from Nonpregnant and Late-Pregnant Rats

In freshly dissociated uterine myocytes, the outward current is carried by K⁺ through channels highly selective for K⁺. Typically, nonpregnant myocytes have rather noisy K⁺ currents; half of them also have a fast-inactivating transient outward current (I_TO). In contrast, the current records are not noisy in late pregnant myocytes, and I_TO densities are low. The whole-cell I_K of nonpregnant myocytes respond strongly to changes in [Ca²⁺]_o or changes in [Ca²⁺]_i caused by photolysis of caged Ca²⁺ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca²⁺ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at −80, −40, or 0 mV and digital subtractions, the whole-cell I_K of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately −61 and −22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca²⁺-activated K⁺ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca²⁺-sensitive K⁺ channels also have a half-open probability at 68 mV, and are less sensitive to Ca²⁺ than similar channels in taenia coli myocytes. Ca²⁺-activated K⁺ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30–35% of the total I_K in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute ∼20% of total I_K in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K⁺ currents that include a suppression of the I_TO, a redistribution of I_K expression from large-conductance Ca²⁺-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca²⁺ sensitivity, and a positive shift of the activation of some large-conductance Ca²⁺-activated channels.     

Digital image analysis of AgNORs in cervical smears of women with premalignant and malignant lesions of the uterine cervix

We performed a hospital-based, unmatched case-control study to investigate the association between progressive stages of cervical neoplasia and digital analysis of cell proliferation by silver stained nucleolus organizer region associated proteins (AgNORs). We measured cell proliferation levels in the cervical epithelial cells of 10 women with low grade squamous intraepithelial lesions (LG-SIL), eight with high grade squamous intraepithelial lesions (HG-SIL), 11 with cervical cancer (CC) and eight with no cervical lesions (controls) using the AgNORs technique. Cell proliferation was measured by digital image analysis (DIA). DIA revealed increased total areas of AgNORs in HG-SIL and CC compared to LG-SIL and control patients. AgNORs with a kidney or cluster shape exhibited greater areas than those with a spherical or long shape. We propose a cut-off of 118 pixels to differentiate benign (control and LG-SIL) from malignant (HG-SIL and CC) lesions. DIA of AgNORs is a simple and inexpensive method for studying proliferation. The increased total area of AgNORs in malignant lesions provides information regarding cell behavior and may be related to cervical carcinogenesis; however, further validation studies are required to establish its usefulness in cytological analysis.     

培养大鼠颈上神经节单烟碱受体通道及突触电流的膜片钳研究

在培养１─２周的大鼠颈上神经节交感神经元标本上,用膜片钳技术记录了单烟碱受体通道电流及胆碱能突触电流,并分析了它们之间的关系。单烟碱受体通道至少有三种亚导状态,即１５ｐＳ,２７ｐＳ,３８ｐＳ,其中以２７ｐＳ最常见。通道有两种开放模式,即单个短促开放与长串开放。对应的平均开放时间分别为τ＿１＝１．７１ｍｓ,τ＿２＝１２．２４ｍｓ。对自发突触电流的分析表明,其下降相的衰减时间常数（τ＝１５．７±１ｍｓ）与上述长串开放的持续时间相当,提示在突触传递过程中,突触前末梢释放的ＡＣｈ引起了突触后神经元烟碱受体通道的长串开放。     

Appearance of retrogradely labeled neurons in the rat superior cervical ganglion after injection of wheat-germ agglutinin-horseradish peroxidase conjugate into the contralateral ganglion

Summary Injection of wheat-germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) into the superior cervical ganglion (SCG) of the rat results in accumulation of WGA-HRP in sympathetic postganglionic neurons in the contralateral SCG. The sympathetic pathways involved and the mechanism underlying the labeling were investigated. The labeling in neurons in the contralateral SCG was apparent 6 h after injection and increased in intensity with longer survival times. The number of labeled neurons reached 1300 at 72 h after the injection. Transection of the external (ECN) or internal carotid nerves (ICN) resulted in considerable reduction in the number of labeled neurons. Combined transection of both ECN and ICN virtually eliminated labeling in the contralateral SCG. This provides strong evidence that these two nerves are the major pathways for WGA-HRP transport out of the SCG. No labeling was observed in the contralateral SCG following injection of horseradish peroxidase (HRP). Therefore, it seems unlikely that a direct nerve connection exists between the bilateral ganglia. Instead, the labeling of contralateral SCG neurons appears to depend on the transneuronal transport capacity of WGA-HRP, which conveys the marker in an anterograde direction along the postganglionic fibers to terminals in sympathetic target organs, and then delivers it transneuronally to contralateral SCG neurons. We suggest that the sympathetic nerve fibers originating in the bilateral SCGs run intermingled and are in close contact in their peripheral target organs.     

Methylation of the Putative Tumor Suppressor Gene RASSF1A in Primary Cervical Tumors

Methylation-sensitive restriction endonuclease analysis (MSRA) followed by polymerase chain reaction (PCR) have been used to estimate the methylation level of 13 CpG dinucleotides in the promoter region of the putative suppressor gene RASSF1A (3p21.31) in squamous cell carcinomas of the uterine cervix (SCCs) carrying human papillomavirus (HPV) types 16, 18, and related types. Methylation of 3 to 13 CpG pairs has been found in 64% (25 out of 39) tumor DNA samples, 22% (2 out of 9) DNA samples from morphologically normal tissues adjacent to the tumor (P = 0.0306), and two out of three DNA samples from peripheral blood leukocytes of carcinoma patients. These CpG pairs are not methylated in the DNA of leukocytes of healthy donors (0 out of 10). The methylation level of the RASSF1A promoter region studied in tumors of the patients with regional lymph node metastases is significantly higher than in tumors of the patient that have no metastases (P = 8.5 × 10^–12). The methylation frequency of gene RASSF1A is two times higher than the frequency of hemi- and homozygous deletions in the chromosome 3 region where the gene is located. The data obtained indicate that methylation is one of the main mechanisms of the RASSF1A gene inactivation in HPV-positive human cervical tumors. The methylation of this gene may be an early event in the genesis of cervical tumors, the methylation level increasing with tumor progression.