This study reports the structure and expression rates of genes of the transforming growth factor-alpha (TGF-α) signal transduction pathway (TGF-α, epidermal growth factor receptor [EGF-R], jun, myc, and metallothionein [MT]) in 47 specimens of ovarian cancer and 21 nonmalignant tissues. The objective was to establish a direct correlation between the genetic activities and the malignant phenotype of the ovarian cancer. The Southern blot technique identified four samples with myc amplification and two with rearranged EGF-R genes. By using the S1 nuclease assay, the analysis of myc transcription showed a similar use of both promotors. Although the size of the investigated transcripts was unaltered, significant differences in the transcription rates were noticed in malignant tissue probes (using northern blot analysis and RNAase protection assay). The following results of messenger RNA analysis in ovarian cancer were observed: EGF-R, negative in 25%, low in 65%, and strongly positive in 10%; TGF-α, negative in 34%, low in 36%, and strongly positive in 30%; myc, negative in 8%, low in 64%, and strongly positive in 28%; jun, negative in 4%, low in 58%, and strong in 38%; and MT, low in 80% and strongly positive in 20%. In most nonmalignant tissues studied, no or only a low expression of TGF-α, EGF-R, and myc. was found. A comparison of these messenger RNA results with the clinical data from tumors showed four different subgroups of ovarian carcinomas. The results of chemotherapy were known in 32 cases. Tumors with negative or low expression rates of all investigated genes did not respond to chemotherapy; 13 of 18 tumors with high expression rates did respond. Additional signal transduction chains distinct from the TGF-α pathway, however, are likely to influence both the expression and activity of transcription factors and MT.
Abstract In developing brain neuronal migration, dendrite outgrowth and dendritic spine outgrowth are controlled by Cdc42, a small GTPase of the Rho family, and its activators. Cdc42 function in promoting actin polymerization is crucial for glutamatergic synapse regulation. Here, we focus on GABAergic synapse-specific activator of Cdc42, collybistin (CB) and examine functional differences between its splice isoforms CB1 and CB2. We report that CB1 and CB2 differentially regulate GABAergic synapse formation in vitro along proximal-distal axis and adult-born neuron maturation in vivo . The functional specialization between CB1 and CB2 isoforms arises from their differential protein half-life, in turn regulated by ubiquitin conjugation of the unique CB1 C-terminus. We report that CB1 and CB2 negatively regulate Cdc42; however, Cdc42 activation is dependent on CB interaction with gephyrin. During hippocampal adult neurogenesis CB1 regulates neuronal migration, while CB2 is essential for dendrite outgrowth. Finally, using mice lacking Gabra2 subunit, we show that CB1 function is downstream of GABA A Rs, and we can rescue adult neurogenesis deficit observed in Gabra2 KO. Overall, our results uncover previously unexpected role for CB isoforms downstream of α2-containing GABA A Rs during neuron maturation in a Cdc42 dependent mechanism. Author Summary GABAergic inhibition regulates distinct stages of brain development; however, cellular mechanisms downstream of GABA A receptors (GABA A Rs) that influence neuronal migration, maturation and synaptogenesis are less clear. ArfGEF9 encodes for RhoGEF with Cdc42 and TC10 GTPase as its substrates. Interestingly, ArhGEF9 is the only known RhoGEF essential for GABAergic synapse formation and maintenance. We report that during brain development ArfGEF9 mRNA splicing regulation generates different ratios of CB1 and CB2 splice isoforms. CB1 mRNA splicing is enhanced during early brain developmental, while CB2 levels remains constant throughout brain development. We also show that CB1 protein has shorter half-life and ubiquitin proteasome system restricts CB1 abundance within developing neuron to modulate neuron migration and distributing GABAergic synapse along the proximal-distal axis. On the other hand, CB2 isoform although expressed abundantly throughout brain development is essential for neuron dendrite maturation. Together, our data identifies specific post-transcriptional and post-translational mechanisms downstream of GABA A Rs influencing ArhGEF9 function to regulate distinct aspects of neuronal maturation process.
The varying tumor-biological behavior of ovarian carcinomas probably influences both their operability and response to chemotherapy, which are the most relevant prognostic factors. The phenotype of different ovarian carcinomas is obviously associated with an activation of the EGF/TGF-alpha signal pathway, including c-myc and c-jun expression. Analysis of EGF-R, TGF-alpha, c-myc and c-jun expression in 33 stage III/IV, and 2 stage I/II ovarian carcinomas with biochemical, molecular-chemical and immunohistochemical methods showed a correlation between the mRNA and protein levels of EGF-R and TGF-alpha for tumors with low or high expressing rates. However, the concentration of measurable free EGF-Rs seems to depend on the amount of TGF-alpha expression by the tumors. The EGF-R binding ligand TGF-alpha is produced by epithelial tumor cells; stromal cells are usually TGF-alpha-negative, as shown by immunohistochemistry. High expression rates of EGF-R. TGF-alpha and c-myc were detected in 6, 7, and 10 out of 35 ovarian carcinomas, respectively. C-jun mRNA was detected in 18/19 cases studied. Non-malignant tissues originating from myometrium or ovary expressed no (or only small amounts of) EGF-R or TGF-alpha mRNA, whereas a high c-myc expression was found in 1/7 normal myometria, and in 2/5 normal ovaries. There was no strong correlation between EGF-R/TGF-alpha and c-myc/c-jun expression.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract The distribution of transforming growth factor α (TGF‐α) in human normal tissues from the uterus, Fallopian tube, ovary, small and large intestine, lung, spleen, kidney, and skin was studied by immunohistochemistry. TGF‐α was found in epidermis, bronchial epithelium, intestinal mucosa, renal tubules, endo‐ as well as in exocervical and endometrial epithelium, and in the serous epithelium of the Fallopian tube. No TGF‐α was detected in the stromal components of any of the tissues nor in any of the pre‐ and post‐menopausal ovaries studied. Twenty‐nine ovarian tumours including 23 ovarian carcinomas, one malignant mixed Muellerian tumour, two ovarian metastases of gastrointestinal carcinomas, one dysgerminoma, one sarcoma, and one fibroma were studied for TGF‐α by the same immunohistochemical method. In 25 cases, specific cytoplasmic staining for TGF‐α of epithelial tumour cells could be demonstrated. The pattern and intensity of the TGF‐α immunostain varied among the TGF‐α‐positive tumours. No TGF‐α was found by immunohistochemistry in the remaining four cases nor in the stromal tumour components of any of the lesions studied. Northern blot analysis for TGF‐α mRNA was performed on 12 of the tumours. While the immunohistochemistry and blotting results correlated well in ten cases, discordant results were obtained in two lesions.
In principle, there are three defined procedures to obtain approval for a medicinal product in the European Union. As discussed in this overview of the procedures, the decision on which regulatory pathway to use will depend on the nature of the active substance, the target indication(s), the history of product and/or the marketing strategy.
The expression of the epidermal growth factor receptor (EGF-R) and the c-myc oncogene was investigated in different specimens of ovarian and cervical carcinomas. The EGF-Rs were analyzed by EGF binding assay, immunohistochemistry and Northern blotting. For analysis of c-myc expression, we used Northern blotting and immunohistochemistry. Furthermore, tissue concentrations of EGF-like factors (EGF-F) were measured in the same tumor and non-malignant specimens. The biochemical determination of EGF-R demonstrated that EGF specific binding sites were detected in 36% of ovarian (n = 140) and 81% of cervical carcinomas (n = 42). High amounts of EGF-R (greater than 10 fmol/mg specific binding) were found in 8% of the ovarian and 41% of the cervical carcinomas. Increased expression of EGF-R specific mRNA was detectable in 7/21 ovarian and in 5/7 cervical carcinomas. A positive correlation between the amounts of EGF-R mRNA, the EGF-R binding data and the staining index of EGF-R immunohistochemistry was found. The EGF-R immunohistochemistry demonstrates that only the tumor cells produce increased amounts of EGF-R, while the stromal cells are EGF-R negative. Low amounts of EGF-R specific mRNA were also detected in biochemically EGF-R negative tumors. The c-myc specific mRNA signal was found in all cases investigated. It is shown that the c-myc expression was increased in 10/21 ovarian and 5/7 cervical carcinomas. There was no positive correlation between the amounts of EGF-R and c-myc mRNAs. The product of myc, as detected by immunohistochemistry, is found in tumor as well as in stromal cells. The levels of EGF-F were measured in extracts of 63 ovarian and 12 cervical carcinomas and in 21 non-malignant tissues. About 30% of the tumor extracts contained higher EGF-F levels (4-15 ng/mg) than those found in the non-malignant specimens. Tumors with high EGF-F levels expressed high amounts of c-myc RNA. The EGF-R status (n = 111) and the EGF-F levels (n = 63) were related to the prognosis of survival for patients with ovarian carcinomas. EGF-R positive (EGF-R(+)) ovarian carcinomas had a significantly higher response rate to chemotherapy. The survival time of the EGF-R(+) group is reduced compared to the EGF-R negative (EGF-R(-)) group if only patients in remission are used to construct survival curves. Furthermore, a poor prognosis for survival was noticed for ovarian carcinoma patients with high EGF-F levels.
