Progesterone receptor expression in medroxyprogesterone acetate-induced murine mammary carcinomas and response to endocrine treatment

Using medroxyprogesterone acetate (MPA) as a carcinogen, we were able to induce in BALB/c female mice, several progestin-dependent mammary ductal carcinomas that regress completely with estrogen or antiprogestins and are maintained by serial transplantations in syngeneic mice. Progestin-independent variants were subsequently generated or appeared spontaneously. Based on their response to estrogen or antiprogestins, we subdivided them into responsive progestin-independent (R-PI) variants which regress completely and unresponsive progestin-independent (UR-PI) carcinomas which are resistant to both families of compounds. In this study we have investigated progesterone receptor (PR) expression in six responsive progestin-dependent, six R-PI, and three UR-PI tumors. Progestin-dependent and R-PI tumors disclosed a higher expression of the PR_A isoform as compared with PR_B, as well as an additional band of 78 kDa that was not detected in uterine tissue; all were down-regulated by progestins. UR-PI tumors expressed lower levels of all bands in western blots, but were highly reactive by immunohistochemistry. PR RNA expression was detected in both, UR-PI and R-PI tumors. PR binding was comparable in progestin-dependent and R-PI tumors. In the three UR-PI tumors, only 29/61 (48%) of the samples evaluated showed low binding levels, the rest were negative. This report is the first to describe in an experimental model of breast cancer the expression of PR isoforms and their distribution. Our results suggest the expression of functionally altered isoforms in a subgroup of mammary carcinomas, which may explain their lack of hormone response.     

Gabaergic signaling mediates the morphological organization of astrocytes in the adult rat forebrain

Previous studies have provided evidence that the morphological organization of immature astrocytes is influenced by the inhibitory neuronal transmitter gamma amino-butyric acid (GABA). The present study was designed to determine whether the occurrence of differential organization of mature astrocytes throughout various regions of the adult brain is related to differential GABAergic signaling. For this we first used Western blotting and high-performance liquid chromatography to quantify the levels of the astrocytic protein glial fibrillary acidic protein (GFAP) and GABA, respectively, within the same tissue punches taken from different forebrain regions of the adult rat, as well as immunocytochemistry for GFAP, GABA, or glutamate decarboxylase to visualize the morphological organization of astrocytes and of GABAergic axons in these regions. These data indicate that GFAP and GABA contents are correlated throughout the different forebrain regions analyzed, and that the regions containing the highest densities in GABAergic terminals are those that contain astrocytes exhibiting the highest degree of stellation. Secondly, we chronically increased GABAergic signaling in vivo by the systemic administration of an inhibitor of GABA transaminase or by the intracerebroventricular infusion of muscimol, a potent agonist of GABA(A) receptors. Our data show that in both cases, the GFAP content of the different forebrain regions is significantly augmented, in close association with a marked increase in the number of astrocytic processes and with their degree of branching. Taken together, these data strongly suggest that GABAergic signaling mediates the morphological organization of astrocytes and their expression of GFAP in the adult brain.     

Regulation of tyrosine hydroxylase levels and activity and Fos expression during opioid withdrawal in the hypothalamic PVN and medulla oblongata catecholaminergic cell groups innervating the PVN

Morphine withdrawal increases the hypothalamic-pituitary-adrenocortical (HPA) axis activity, which is dependent on an hyperactivity of noradrenergic pathways innervating the hypothalamic paraventricular nucleus (PVN). However, the possible adaptive changes that can occur in these pathways during morphine dependence are not known. We studied the alterations in tyrosine hydroxylase (TH; the rate-limiting enzyme in catecholamines biosynthesis) immunoreactivity levels and TH enzyme activity in the rat NTS-A2/VLM-A1 noradrenergic cell groups and in the PVN during morphine withdrawal. In the same paradigm, we measured Fos expression as a marker of neuronal activation. TH and Fos immunoreactivity was determined by quantitative Western blot analysis, combined with immunostaining for TH and Fos for immunohistochemical identification of active neurons during morphine withdrawal. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (5 mg/kg s.c.). Morphine withdrawal induced the expression of Fos in the PVN and NTS/VLM, which indicates an activation of neurons in these nuclei. TH immunoreactivity in the NTS/VLM was increased 90 min after morphine withdrawal, whereas there was a decrease in TH levels in the PVN at the same time point. Following withdrawal, Fos immunoreactivity was present in most of the TH-positive neurons of the A2 and A1 neurons. TH activity was measured in the PVN, a projection area of noradrenergic neurons arising from NTS-A2/VLM-A1. Morphine withdrawal was associated with an increase in the enzyme activity at different time points after naloxone-precipitated morphine withdrawal. The present results suggest that an increase in TH protein levels and TH enzyme activity might contribute to the enhanced noradrenergic activity in the PVN in response to morphine withdrawal.     

Absence of rearrangements in the BRCA2 gene in human cancers

Mutations of BRCA2 in sporadic breast and ovarian carcinomas are exceedingly rare. This led to the suggestion that large genomic rearrangements could be involved. We performed Southern blots in genomic DNA from 130 primary breast cancers and 83 cancer cell lines (breast, ovarian, pancreatic and small cell lung carcinomas) and found no genomic rearrangements. These results suggest that a gene other than BRCA2 is the target of the frequent 13q12.3 allelic deletions in human cancers.     

Multidrug resistance gene (MDR-1) expression as a useful prognostic factor in patients with human hepatocellular carcinoma after surgical resection

BACKGROUND: Multidrug resistance gene (MDR-1) overexpression has been correlated with tumor aggressiveness and worse prognosis in some human neoplasms. The aim of this study is to evaluate the clinical value of MDR-1 mRNA expression as a prognostic factor after surgical resection in human hepatocellular carcinoma (HCC). METHODS: MDR-1 mRNA levels in tissue samples from 34 patients with HCC, who underwent surgical resection, were measured by quantitative northern blot analysis. We stratified these patients into two groups according to a ratio of MDR-1 mRNA levels of HCC to nontumorous tissue; MDR-1 mRNA ratio > or = 1.0 and < 1.0. The overall and disease-free survival rates were analyzed using multivariate regression analysis. RESULTS: The median survival periods were 10.3 and 35.8 months for patients with the MDR-1 mRNA ratio > or = 1.0 and < 1.0, respectively, and the corresponding 5-year survival rates were 33 and 54%, respectively, P < 0.05. The multivariate analysis revealed that TNM stage and MDR-1 mRNA ratio were independent factors for predicting overall survival after surgical resection. CONCLUSION: This study suggested that the measurement of the MDR-1 mRNA levels in HCC and nontumorous liver tissue might be a useful prognostic factor after surgical resection in patients with HCC.     

Neuronal and glial beta-secretase (BACE) protein expression in transgenic Tg2576 mice with amyloid plaque pathology

We measured tissue distribution and expression pattern of the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE) in the brains of transgenic Tg2576 mice that show amyloid pathology. BACE protein was expressed at high levels in brain; at lower levels in heart and liver; and at very low levels in pancreas, kidney, and thymus and was almost absent in spleen and lung when assayed by Western blot analysis. We observed strictly neuronal expression of BACE protein in the brains of nontransgenic control mice, with the most robust immunocytochemical labeling present in the cerebral cortex, hippocampal formation, thalamus, and cholinergic basal forebrain nuclei. BACE protein levels did not differ significantly between control and transgenic mice or as a result of aging. However, in the aged, 17-month-old Tg2576 mice there was robust amyloid plaque formation, and BACE protein was also present in reactive astrocytes present near amyloid plaques, as shown by double immunofluorescent labeling and confocal laser scanning microscopy. The lack of astrocytic BACE immunoreactivity in young transgenic Tg2576 mice suggests that it is not the APP overexpression but rather the amyloid plaque formation that stimulates astrocytic BACE expression in Tg2576 mice. Our data also suggest that the neuronal overexpression of APP does not induce the overexpression of its metabolizing enzyme in neurons. Alternatively, the age-dependent accumulation of amyloid plaques in the Tg2576 mice does not require increased neuronal expression of BACE. Our data support the hypothesis that neurons are the primary source of beta-amyloid peptides in brain and that astrocytic beta-amyloid generation may contribute to amyloid plaque formation at later stages or under conditions when astrocytes are activated.     

Design and validation of immunological tests for the detection of Porcine endogenous retrovirus in biological materials

The present study details the design and demonstrates function for a series of reagents and methods to allow the detection of exposure to antigens specific for Porcine endogenous retrovirus (PERV). The detection of PERV is carried out by the means of a variety of immunological screening methods including, indirect immunofluorescence, Western blotting and enzyme linked immunosorbent assay (ELISA) for the detection of antibodies in serum specific for PERV gag and env antigens. Alternatively, PERV-specific antisera for gag and env can be used to detect viral antigen in serum or other samples. PERV env peptides with potential specificity for the known PERV types are also described. Antisera against the peptides can be used to detect PERV antigens directly or to characterise viral type. Using electron microscopy coupled with labelled PERV-gag-specific antisera it was possible to visualise PERV virions.     

Not the presence but the amount of clonal DNA detectable in remission of acute myeloid leukemia is predictive for relapse

Abstract: Objectives: The persistence of clonal cells after chemotherapy, or a re‐emerging of clonal cells in remission (CR) or at relapse in patients with acute myeloid leukemia (AML) was studied to assess the prognostic significance of the amount of clonal DNA in predicting the clinical outcome. Methods: Clonal rearrangements in the gene sequences of retinoic acid receptor (RAR) α, major breakpoint cluster region (M‐bcr), immunoglobulin (Ig)‐JH, T‐cell receptor (TcR) β, myeloid lymphoid leukemia or cytokines (GM‐CSF, G‐CSF, IL‐3) detected in bone marrow samples from 37 patients with primary AML (pAML) or secondary AML (sAML) were investigated. A relative increase or decrease of clonal DNA in the course of AML was evaluated by comparing the optical densities of DNA bands of the rearranged genes and the total amount of DNA. Results: High amounts of clonal DNA were detectable at diagnosis, during persisting disease and at relapse (Ø 39%, 35%, or 38% of total DNA, respectively), compared to 20% in complete remission (CR). Amounts of clonal DNA (except for Ig‐JH gene rearrangements) were of prognostic significance at diagnosis, patients with less than 33% clonal DNA were characterized by significantly longer relapse‐free survival times (all cases: p = 0.01; pAML: p = 0.002). Patients in CR exhibiting less than 5% (all cases) or 15% (pAML) clonal DNA showed longer relapse‐free survival times (p = 0.08 or p = 0.03, respectively). Vice versa, significantly higher amounts of clonal DNA (all cases 51% vs. pAML 54%) could be detected in cases studied at diagnosis who relapsed in the following 5 months (all cases p = 0.01) or 14 months (pAML p = 0.007). Significantly higher amounts of clonal DNA (33%) could be detected in cases studied in CR who relapsed in the following 4 months (all cases p = 0.002 or pAML p = 0.006, respectively). Moreover, we could prove disease progression on a cellular level months before the clinical onset of sAML after a period of MDS. Conclusions: Clonal, gene‐rearranged DNA is regularly detectable at diagnosis and during persisting AML, in CR and at relapse. However, the presence, rather than the amount of clonal DNA detectable in CR is predictive for relapse. These data might indicate the significance of gene rearrangement analyses in the course of AML to identify cases with a high risk of relapse, independently from the karyotype.     

应用寡核苷酸探针阵列法快速鉴定临床分离株中的分支杆菌

目的:建立一种简便、快速、灵敏、特异的分支杆菌菌种鉴定方法。方法:通过16S rRNA聚合酶链反应(polym erase chain reaction,PCR)-单链构象多态性(single-stranded conform ation polymorph ism,SSCP)分析鉴定253株分支杆菌临床分离株;应用16S rRNA PCR-寡核苷酸探针与待测菌株生物素标记的16S rRNA基因PCR产物进行反向斑点杂交。结果:分析28种分支杆菌标准菌株和9种非分支杆菌菌株,结果显示寡核苷酸探针是特异的。253株分支杆菌临床分离株,198株为结核分支杆菌复合群,55株为非结核分支杆菌。经寡核苷酸探针阵列法分析,198株结核分支杆菌分离株鉴定为结核分支杆菌复合群,36株鉴定为非结核分支杆菌,分别与相对应的特异探针杂交,另19株与分析探针杂交阴性。结论:16S rRNA PCR-寡核苷酸探针阵列法灵敏度高、特异性强、简便、快速,可用于鉴定临床分离株分支杆菌菌种。     

丝裂原活化蛋白激酶在卵巢癌中的表达及意义

目的:探讨丝裂原活化蛋白激酶(MAPKs)的三个亚族ERK1J、NK1、p38在卵巢癌中表达的意义及三者之间的关系。方法:应用免疫组化及Western bloting印迹分析的方法,检测ERK1、JNKp、38在50例卵巢上皮癌、30例卵巢良性上皮性肿瘤和30例正常卵巢组织中的表达,判断ERK1J、NK1、p38与临床病理因素的关系,明确MAPKs亚族间的相关性。结果:JNK1、p38在Ⅰ~Ⅱ期卵巢癌组织中的表达明显高于正常卵巢组织和卵巢良性上皮性肿瘤(P<0.05)。ERK1在Ⅲ~Ⅳ期卵巢癌组织中的表达明显高于正常卵巢组织和卵巢良性上皮性肿瘤(P<0.05)。JNK1与p38蛋白阳性率呈明显的正相关(P<0.05)。结论:MAPKs不同亚族在卵巢癌的发病学中起到重要作用,并与卵巢癌期别有关。