56 research outputs found
17-β-Estradiol-dependent regulation of somatostatin receptor subtype expression in the 7315b prolactin secreting rat pituitary tumor in vitro and in vivo
In the present study, we have investigated the role of estrogens in the regulation of somatostatin receptor subtype (sst) expression in 7315b PRL- secreting rat pituitary tumor cells in vitro and in vivo. sst were undetectable in freshly dispersed cells of the transplantable 7315b tumor. When 7315b cells were cultured in medium containing 10% FCS, the number of high affinity sst increased with prolonged culture time. However, when the medium was supplemented with 10% horse serum (HS) instead of FCS, no sst were detectable on 7315b cells even after three weeks of culturing. In contrast to HS, FCS contains high E2-levels (HS, 8 pM; FCS, 134 pM). The antiestrogen tamoxifen (0.5 ÎźM) significantly inhibited the sst number to 50.5% of the value of untreated FCS-grown cells, suggesting that E2 stimulates sst expression in 7315b rat pituitary tumor cells. E2 (l0 nM) induced a rapid increase in sst number in HS-grown 7315b cells. Octreotide (1ÎźM) significantly inhibited PRL release and the intracellular PRL concentration of 7315b cells that were cultured in medium supplemented with FCS or with HS + l0 nM E2 but not in HS alone. This indicates that the sst present on these cells are biologically active. RT-PCR analysis revealed that none of the five currently known sst subtypes were present in freshly dispersed 7315b pituitary tumor cells. The expression of sst2- and sst3- messenger RNA (mRNA) was unequivocally correlated to the presence of E2 because these sst subtypes were detected only in cells that were cultured for7 and 14 days in medium supplemented with FCS or with HS + 10 nM E2. sst1, sst4 and sst5 messenger RNA could not be detected. The 7315b tumor itself synthesizes and secretes huge amounts of PRL. The high PRL levels in tumor-bearing rats inhibit the ovarian E2-production. No detectable E2 levels could be measured in the serum of 7315b tumor-bearing rats. The sc administration of 20 Îźg/day E2-benzoate normalized the circulating E2 levels in 7315b tumor- bearing rats. Moreover, E2-treatment indeed induced sst expression in vivo as shown by ligand binding studies using membrane homogenates and [125I- Tyr3]-octreotide as radioligand and by autoradiography on tissue sections. In agreement with the in vitro studies, the expression of the sst2 subtype was established by RT-PCR analysis in 7315b tumors of E2-treated rats. However, in contrast to the in vitro studies. E2-treatment did not effectuate the expression of the sst3 subtype, suggesting that the in vitro stimulus of E2 is stronger. In conclusion: 1) sst2 and sst3 expression in the 7315b rat prolactinoma model is primarily dependent upon the presence of estrogens; 2) the antihormonal action of octreotide in 7315b tumor cells in vitro is mediated via the sst2 and/or sst3 subtypes; 3) the absence of sst expression in vivo can be explained by the hormonal environment of the 7315b tumor cells. The 7315b tumor cells in vivo may down-regulate their own receptor status via their host, because of the ensuing hyperprolactinemia results in a hypo-estrogenic state.</p
Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9
CRISPR/Cas9 technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells. Similarly, certain endogenous genes have been tagged with fluorescent proteins. Often, the detection of tagged proteins requires high expression and sophisticated tools such as confocal microscopy and flow cytometry. Therefore, a simple, sensitive and robust transcriptional reporter system driven by endogenous promoter for studies into transcriptional regulation is desirable. We report a CRISPR/Cas9-based methodology for rapidly integrating a firefly luciferase gene in somatic cells under the control of endogenous promoter, using the TGFβ-responsive gene PAI-1. Our strategy employed a polycistronic cassette containing a non-fused GFP protein to ensure the detection of transgene delivery and rapid isolation of positive clones. We demonstrate that firefly luciferase cDNA can be efficiently delivered downstream of the promoter of the TGFβ-responsive gene PAI-1. Using chemical and genetic regulators of TGFβ signalling, we show that it mimics the transcriptional regulation of endogenous PAI-1 expression. Our unique approach has the potential to expedite studies on transcription of any gene in the context of its native chromatin landscape in somatic cells, allowing for robust high-throughput chemical and genetic screens
A potential tool in the battle against gastroenteropancreatic neuroendocrine tumors: Interferon-beta
Daphnid life cycle response to new generation of flame retardants
Relatively hazardous brominated flame retardants (BFRs) are currently substituted with halogen-free flame retardants (HFFRs). Consequently, information on their persistence, bioaccumulation and toxicity (PBT) is urgently needed. Therefore, we investigated the chronic toxicity to the water flea Daphnia magna of two HFFRs, aluminum diethylphosphinate (ALPI) and 9,10-dihyro-9-oxa-10-phosphaphenanthrene-oxide (DOPO). The toxicity of ALPI increased from a 48 h LC50 of 18 mg L-1 to a 21 day LC50 value of 3.2 mg L-1, resulting in an acute-to-chronic ratio of 5.6. This may imply a change in classification from low to moderate toxicity. ALPI also affected sublethal life cycle parameters, with an EC50 of 2.8 mg L-1 for cumulative reproductive output and of 3.4 mg L-1 for population growth rate, revealing a nonspecific mode of action. DOPO showed only sublethal effects with an EC50 value of 48 mg L-1 for cumulative reproductive output and an EC50 value of 73 mg L-1 for population growth rate. The toxicity of DOPO to D. magna was classified as low and likely occurred above environmentally relevant concentrations, but we identified specific effects on reproduction. Given the low chronic toxicity of DOPO and the moderate toxicity of ALPI, based on this study only, DOPO seems to be more suitable than ALPI for BFR replacement in polymers
Longâterm inâvitro treatment of human growth hormone (GH)âsecreting pituitary adenoma cells with octreotide causes accumulation of intracellular GH and GH mRNA levels
OBJECTIVE We studied the effects of longâterm inâvitro exposure of human GH secreting pituitary adenoma cells to octreotide on GH release, intracellular GH concentrations and GH messengerbonuclelc acid (mRNA) levels. DESIGN Human GHâsecreting pituitary adenoma cells were cultured for periods from 4 days up to 3 weeks without or with octreotide (10 PM) and/or bromocriptine (10 nM). The effects of these drugs were measured on GH release, intracellular GH concentrations and intracellular GH mRNA levels. PATIENTS Thirteen patients with GHâsecreting pituitary adenomas were studied. Twelve patients were untreated, one had been pretreated with octreotide (12 weeks, 3 Âą 100 Îźg daily). MEASUREMENTS GH, PRL, Îąâsubunlt and IGFâI concentrations in plasma, media and cell extracts were determined by Immunoradiometric or radloimmunoassays. GH mRNA levels were determined by automatic quantification of grain numbers in individual adenoma cells. RESULTS Incubation of the adenoma cells for 4 days with 10 Nm octreotide induced a doseâdependent inhibition of GH release and a parallel increase (increase varying between 124 and 617% of control) In the intracellular GH levels was observed in six of seven adenomas. In addition, bromocriptine, when effective in inhibiting GH release by the adenomas, also induced an increase in intracellular GH levels. Even after 3 weeks of exposure to 10 nM octreotide in vitro there was a statistically significant increase in intracellular GH levels (between 191 and 923% of control). Withdrawal of octreotide after 6 days of incubation resulted in a lowering of intracellular GH levels to control values, showing that the octreotideâinduced increase in intracellular GH is reversible. In a 96âhour incubation with 10 nM octreotide, GH mRNA levels were increased in two, and slightly decreased in one of the three adenomas tested. This effect was time dependent in that there was no significant effect of 10 nM octreotide on GH mRNA levels in a 24âhour incubation. CONCLUSIONS (1) Longâterm inâvitro exposure of GHâadenoma cells to octreotide causes an increase in intracellular GH levels in the majority of the adenomas, probably because of an increase in GH mRNA levels in the adenoma cells; and (2) this considerable increase in intracellular GH levels may be one of the explanations for the relatively poor effect of octreotide on tumour shrinkage In patients with GHâsecreting pituitary adenomas.</p
Daphnid Life Cycle Responses to New Generation Flame Retardants
Relatively hazardous brominated flame retardants (BFRs) are currently substituted with halogen-free flame retardants (HFFRs). Consequently, information on their persistence, bioaccumulation and toxicity (PBT) is urgently needed. Therefore, we investigated the chronic toxicity to the water flea Daphnia magna of two HFFRs, aluminum diethylphosphinate (ALPI) and 9,10-dihyro-9-oxa-10-phosphaphenanthrene-oxide (DOPO). The toxicity of ALPI increased from a 48 h LC50 of 18 mg L-1 to a 21 day LC50 value of 3.2 mg L-1, resulting in an acute-to-chronic ratio of 5.6. This may imply a change in classification from low to moderate toxicity. ALPI also affected sublethal life cycle parameters, with an EC50 of 2.8 mg L-1 for cumulative reproductive output and of 3.4 mg L-1 for population growth rate, revealing a nonspecific mode of action. DOPO showed only sublethal effects with an EC50 value of 48 mg L-1 for cumulative reproductive output and an EC50 value of 73 mg L-1 for population growth rate. The toxicity of DOPO to D. magna was classified as low and likely occurred above environmentally relevant concentrations, but we identified specific effects on reproduction. Given the low chronic toxicity of DOPO and the moderate toxicity of ALPI, based on this study only, DOPO seems to be more suitable than ALPI for BFR replacement in polymers
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