Expression Analysis of Genes Involved in the RB/E2F Pathway in Astrocytic Tumors

Astrocytic gliomas, which are derived from glial cells, are considered the most common primary neoplasias of the central nervous system (CNS) and are histologically classified as low grade (I and II) or high grade (III and IV). Recent studies have shown that astrocytoma formation is the result of the deregulation of several pathways, including the RB/E2F pathway, which is commonly deregulated in various human cancers via genetic or epigenetic mechanisms. On the basis of the assumption that the study of the mechanisms controlling the INK4/ARF locus can help elucidate the molecular pathogenesis of astrocytic tumors, identify diagnostic and prognostic markers, and help select appropriate clinical treatments, the present study aimed to evaluate and compare methylation patterns using bisulfite sequencing PCR and evaluate the gene expression profile using real-time PCR in the genes CDKN2A, CDKN2B, CDC6, Bmi-1, CCND1, and RB1 in astrocytic tumors. Our results indicate that all the evaluated genes are not methylated independent of the tumor grade. However, the real-time PCR results indicate that these genes undergo progressive deregulation as a function of the tumor grade. In addition, the genes CDKN2A, CDKN2B, and RB1 were underexpressed, whereas CDC6, Bmi-1, and CCND1 were overexpressed; the increase in gene expression was significantly associated with decreased patient survival. Therefore, we propose that the evaluation of the expression levels of the genes involved in the RB/E2F pathway can be used in the monitoring of patients with astrocytomas in clinical practice and for the prognostic indication of disease progression.     

裸鼠肿瘤动物模型VEGF受体表达及其意义

目的　通过免疫组织化学染色了解flt 1与flk 1 KDR(VEGF的两个高亲和受体 )在人肿瘤细胞皮下接种肿瘤动物模型的血管内皮细胞与肿瘤细胞中的表达。方法　取荷瘤裸鼠皮下接种瘤块 ,漂洗、固定、石蜡连续切片 ,进行两种受体相应免疫组化检测。结果　在 13种荷瘤裸鼠血管内皮细胞及肿瘤细胞中flt 1的阳性率大部分为强阳性或中阳性 ,而只有在荷人胃腺癌MKN 4 5裸鼠的肿瘤细胞中flt 1的阳性率为弱阳性 ,在荷人卵巢癌SKOv3裸鼠的肿瘤细胞中flt 1的表达为阴性。相比较而言 ,在 13种荷瘤裸鼠血管内皮细胞及肿瘤细胞中KDR的阳性率大部分为中阳性或弱阳性 ,并且在荷人肝癌SMMC 772 1裸鼠 ,荷人胃腺癌SPC A1裸鼠 ,荷人高转移肝癌移植瘤裸鼠 ,荷人卵巢癌SKOv3裸鼠的肿瘤细胞中 ,荷人宫颈癌移植瘤裸鼠和荷人胃腺癌MKN 4 5裸鼠的肿瘤细胞中 ,KDR表达为阴性。结论　VEGF受体共同表达于肿瘤血管内皮细胞与肿瘤细胞 ,提示了VEGF与VEGF受体结合作用在肿瘤演化中的重要性 ,为靶向于VEGF受体的基因治疗策略选择裸鼠动物模型提供了参考依据     

Metabolism of prostaglandins E2 and F2 alpha by human fetal membranes.

Prostaglandin E2 (PGE2) is important in the early stages of human labour, leading particularly to cervical ripening and dilatation. The source of PGE2 is thought to be either the amnion or the decidua, but the chorion interposes between the amnion and the target tissues, namely the myometrium and cervix. In order to investigate the role of the chorion in modulating prostanoid production, [3H]PGE2 was added to the amnion side of fetal membranes, and the production of metabolites on both sides of the fetal membrane followed by HPLC. The major metabolite was 13,14-dihydro-15-oxo-PGE2 with smaller amounts of 13,14-dihydro-15-oxo-PGA2 and PGB2. The production of all metabolites of PGE2 was time dependent. [3H]PGF2 alpha, which is normally produced by the decidua, was also added to fetal membranes and found to be metabolised to 13,14-dihydro-15-oxo-PGF2 alpha and PGE2. These results suggest that the metabolic enzymes in the chorion may determine intra-uterine levels of prostaglandins, and may also determine the identity of the eicosanoids released by intact fetal membranes.     

Prostaglandin E2 and F2 alpha in mid-pregnant rat uterus and at parturition

Prostaglandin E2 (PGE2) and F2 alpha (PGF2 alpha) produced by 15 days pregnant rat myometrium, by parturient rat myometrium and myometrium plus endometrium were measured in vitro. The results showed that the PGs produced by parturient myometrium were higher than these obtained during mid-pregnancy. Myometrium with endometrium released more PGs than myometrium alone, and the addition of arachidonic acid (AA) at 10 DM did not show any significant effect. Exogenous progesterone or estradiol-17b at a concentration of 1 Dmol had no effect on parturient uterine PG secretions.     

Expression of enzymes of cyclooxygenase pathway and secretion of prostaglandin E2 and F2alpha by porcine myometrium during luteolysis and early pregnancy

Past studies of uterine prostaglandin (PGs) and pig reproduction have focused on endometrial rather than myometrial PGs. This study documents the synthesis and secretion of myometrial prostaglandins (PGs) in pigs and the involvement of oxytocin (OT) in these processes. Cyclooxygenase-2 (COX-2) expression was similar in myometrial explants from cyclic and pregnant pigs (days 14-16) and OT (10(-7) M) in vitro significantly increased COX-2 protein regardless of reproductive state. Basal expression of prostaglandin E2 synthase (PGES) was higher during pregnancy than during luteolysis. Conversely, prostaglandin F synthase (PGFS) was highest during luteolysis and lower in myometrium from gravid animals. OT had no influence on the expression of PGES and PGFS. In another tissue culture experiment, myometrial slices produced more PGE2 than PGF2alpha regardless of reproductive state of the female. OT stimulated PGE2 production in myometrium harvested during luteolysis and increased PGF2alpha production in all tissues examined. Progesterone (P4; 10(-5) M) blocked stimulatory effect of OT on myometrial PG release. Myometrial OTr mRNA was higher (P=0.03) during luteolysis than during pregnancy. In conclusion: (1) oxytocin increases myometrial COX-2 expression, but does not influence the expression of terminal enzymes of PGs synthesis (PGES and PGFS); (2) porcine myometrium preferentially produces PGs during early pregnancy and secretes more PGE2 than PGF2alpha; (3) myometrial OT and OTr support secretion of PGs from myometrium during luteolysis.     

Effects of prostaglandins E2 and F2 alpha on progesterone metabolism by rat granulosa cells

Rat granulosa cells were cultured with or without PGE2 and/or PGF2 alpha. Accumulation of endogenous progesterone and 20 alpha-hydroxy-4-pregnen-3-one was determined. Additionally, [4-14C]progesterone metabolism was assessed. PGE2 increased progesterone accumulation, in part, by decreasing progesterone catabolism to 20 alpha-reduced progestins. In contrast, PGF2 alpha stimulated 20 alpha-hydroxysteroid dehydrogenase activity, thus increasing progesterone catabolism. Combined treatment with PGE2 and PGF2 alpha augmented progesterone accumulation to levels above controls but below those attained with PGE2 alone. These data indicate that PGE2 and PGF2 alpha exert opposite effects on progesterone production and catabolism and that the ratio of PGE2 to PGF2 alpha in the local granulosa cell milieu may be of importance in determining overall progesterone output.     

E2F activity is biphasically regulated by androgens in LNCaP cells

Androgens exert a peculiar biphasic dose-dependent influence on the proliferation of LNCaP cells, a widely used model to study androgen effects on prostate cancer cells. Low concentrations of androgen stimulate proliferation, but high concentrations inhibit proliferation and induce strong expression of differentiation markers. In order to gain more insight into the molecular mechanisms that underlie these changes we studied the influence of a wide concentration range of the synthetic androgen R1881 on several cell cycle- and differentiation-related parameters. Low concentrations (0.1 nM), known to promote LNCaP cell proliferation, induce an increase of Retinoblastoma protein phosphorylation, accompanied by an increase of E2F-1 protein levels and E2F activity and by increased expression of the E2F-target gene products E2F-1 and cyclin A. High concentrations of R1881 (10 nM) induce strong expression of the differentiation marker prostate-specific antigen. Retinoblastoma protein is largely hypophosphorylated, resulting in low E2F activity and low concentrations of E2F-1 and cyclin A mRNA. Finally, there is a strong increase of p27(KIP1) protein, but not of p27(KIP1) mRNA. These results indicate that the biphasic dose response of LNCaP proliferation to androgen is closely reflected in Rb phosphorylation, E2F activity and p27(KIP1) protein expression.     

E2F7 and E2F8 keep the E2F family in balance

An article by Li and colleagues (in this issue of Developmental Cell) shows that the atypical E2Fs, E2F7 and E2F8, are critical for mouse development. One of the important functions of these family members stems from a negative feedback loop in which E2F7 and E2F8 limit the expression of E2F1 and prevent E2F1-dependent apoptosis.     

Urinary excretion of prostaglandin E2 and prostaglandin F2alpha in potassium-deficient rats

Potassium-deficiency was induced in rats by dietary deprivation of potassium. The animals became polyuric and urine osmolality decreased more then three-fold compared to controls. Urinary excretion of prostaglandin E2 (PGE2) and prostaglandin F2alpha (PGF2alpha) did not increase during 2 weeks of potassium depletion. Partial inhibition of renal prostaglandin synthesis by meclofenamate did not increase the urine osmolality after water deprivation. These results make unlikely the hypothesis that the polyuria of potassium-deficiency, is the result of enhanced renal synthesis of prostaglandins with subsequent antagonism of the hydro-osmotic effect of vasopressin. Male animals consistently excreted less PGE2 than female animals.     

Effect of prostaglandins E1, E2 and F2alpha on neutrophil aggregation.

Recently we have found that chemotactic factors stimulate neutrophils in suspension to aggregate. Because of an obvious analogy to platelet aggregation, we examined the influence of three prostaglandins on this process. Prostaglandins E1, E2 and F2alpha alone did not cause aggregation of the neutrophils but were able to partially inhibit the aggregation response induced by the synthetic chemotactic tripeptide, formyl-methionyl-leucyl-phenylalanine. The minimal inhibitory concentrations for prostaglandins E1, E2 and F2alpha were 10(-7), 10(-6) and 10(-5)M, respectively. These results are similar to those found for the prostaglandin-induced inhibition of platelet aggregation. It may be, therefore, that neutrophil aggregation, like platelet aggregation, is modulated by intracellular prostaglandins and other products of arachidonic acid metabolism.