RESUMO
The radiation exposure of the hands of nuclear medicine laboratory technicians is largely due to the dispensing of radiopharmaceuticals into syringes. To reduce this exposure, a multiradionuclide automatic dispensing system (ADS) for syringes of radiopharmaceuticals was introduced. The aim of this study was to determine the effect of this ADS on hand dose compared with manual dispensing. Methods: The total hand dose per month for all personnel (12 technicians) was measured with ring dosimeters at the base of the index finger for 13 mo: 7 mo with manual syringe dispensing (radiopharmaceuticals containing 99mTc,18F, 177Lu, 68Ga, 90Y, and 223Ra) and 6 mo with ADS (automatic: radiopharmaceuticals containing 18F and 177Lu; manual: radiopharmaceuticals containing 99mTc, 68Ga, 90Y, and 223Ra). Results: The mean total hand dose per month was reduced from 52.8 ± 10.2 mSv with manual dispensing to 21.9 ± 2.7 mSv with ADS (P < 0.001), which is an absolute decrease of 59%. Meanwhile, the total handled activity increased from 369 to 505 GBq (P < 0.001). 18F-containing radiopharmaceuticals were the most commonly dispensed, at 182 GBq per month. The increase in total handled activity was largely due to an increase in 177Lu (from 25 to 123 GBq), partially because of the introduction of [177Lu]Lu-PSMA-I&T. When correcting for this increase in handled activity, the hand dose was reduced by 69%. Conclusion: The introduction of a multiradionuclide syringe ADS decreased the hand dose to personnel by 69% when corrected for the increase in handled activity. Expanding the number of radiopharmaceuticals being dispensed by the system could potentially further decrease personnel hand dose.
RESUMO
The aim of this study was to estimate and subsequently measure the occupational radiation exposure for all personnel involved in producing, administering, or performing imaging or surgery with [99mTc]Tc-PSMA-I&S, which has been introduced for identification of tumor-positive lymph nodes during salvage prostate cancer surgery. Methods: The effective dose was estimated and subsequently measured with electronic personal dosimeters for the following procedures and personnel: labeling and quality control by the radiopharmacy technologist, syringe preparation by the nuclear medicine laboratory technologist, patient administration by the nuclear medicine physician, patient imaging by the nuclear medicine imaging technologist, and robot-assisted laparoscopic salvage lymph node dissection attended by an anesthesiology technologist, scrub nurse, surgical nurse, and surgeon. The dose rate of the patient was measured immediately after administration of [99mTc]Tc-PSMA-I&S, after imaging, and after surgery. Results: The estimated dose per procedure ranged from 1.59 × 10-10 µSv (imaging technologist) to 9.74 µSv (scrub nurse). The measured effective dose ranged from 0 to 5 µSv for all personnel during a single procedure with [99mTc]Tc-PSMA-I&S. The highest effective dose was received by the scrub nurse (3.2 ± 1.3 µSv), whereas the lowest dose was measured for the surgical nurse (0.2 ± 0.5 µSv). If a single scrub nurse were to perform as many as 100 procedures with [99mTc]Tc-PSMA-I&S in a year, the total effective dose would be 320 µSv/y. Immediately after administration, the dose rate at 50 cm from the patient was 18.5 ± 1.6 µSv/h, which dropped to 1.8 ± 0.3 µSv/h after imaging the following day, reducing even further to 0.56 ± 0.33 µSv/h after surgery. Conclusion: The effective dose for personnel involved in handling [99mTc]Tc-PSMA-I&S is comparable to that of other 99mTc-radiopharmaceuticals and therefore safe for imaging and radioguided surgery.
Assuntos
Medicina Nuclear , Exposição Ocupacional , Neoplasias da Próstata , Exposição à Radiação , Lesões por Radiação , Humanos , Masculino , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/cirurgia , Compostos RadiofarmacêuticosRESUMO
With current techniques, it remains a challenge to assess coregulator binding of nuclear receptors, for example, the estrogen receptor alpha (ERα). ERα is critical in many breast tumors and is inhibited by antiestrogens such as tamoxifen in cancer therapy. ERα is also modified by acetylation and phosphorylation that affect responses to the antiestrogens as well as interactions with coregulators. Phosphorylation of ERα at Ser305 is one of the mechanisms causing tamoxifen resistance. Detection of resistance in patient samples would greatly facilitate clinical decisions on treatment, in which such patients would receive other treatments such as aromatase inhibitors or fulvestrant. Here we describe a coregulator peptide array that can be used for high-throughput analysis of full-length estrogen receptor binding. The peptide chip can detect ERα binding in cell and tumor lysates. We show that ERα phosphorylated at Ser305 associates stronger to various coregulator peptides on the chip. This implies that ERαSer305 phosphorylation increases estrogen receptor function. As this is also detected in a breast tumor sample of a tamoxifen-insensitive patient, the peptide array, as described here, may be applicable to detect tamoxifen resistance in breast tumor samples at an early stage of disease and contribute to personalized medicine.
Assuntos
Antineoplásicos Hormonais/farmacologia , Receptor alfa de Estrogênio/metabolismo , Serina/metabolismo , Tamoxifeno/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/genética , Humanos , Análise em Microsséries , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Osteossarcoma/metabolismo , Fosforilação , TransfecçãoRESUMO
Anti-estrogen resistance is a major clinical problem in the treatment of breast cancer. In this study, fluorescence resonance energy transfer (FRET) analysis, a rapid and direct way to monitor conformational changes of estrogen receptor alpha (ERalpha) upon anti-estrogen binding, was used to characterize resistance to anti-estrogens. Nine different anti-estrogens all induced a rapid FRET response within minutes after the compounds have liganded to ERalpha in live cells, corresponding to an inactive conformation of the ERalpha. Phosphorylation of Ser(305) and/or Ser(236) of ERalpha by protein kinase A (PKA) and of Ser(118) by mitogen-activated protein kinase (MAPK) influenced the FRET response differently for the various anti-estrogens. PKA and MAPK are both associated with resistance to anti-estrogens in breast cancer patients. Their respective actions can result in seven different combinations of phospho-modifications in ERalpha where the FRET effects of particular anti-estrogen(s) are nullified. The FRET response provided information on the activity of ERalpha under the various anti-estrogen conditions as measured in a traditional reporter assay. Tamoxifen and EM-652 were the most sensitive to kinase activities, whereas ICI-182,780 (Fulvestrant) and ICI-164,384 were the most stringent. The different responses of anti-estrogens to the various combinations of phospho-modifications in ERalpha elucidate why certain anti-estrogens are more prone than others to develop resistance. These data provide new insights into the mechanism of action of anti-hormones and are critical for selection of the correct individual patient-based endocrine therapy in breast cancer.
Assuntos
Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/fisiologia , Estrogênios/química , Transferência Ressonante de Energia de Fluorescência/métodos , Mutação , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/química , Fulvestranto , Humanos , Proteínas Luminescentes/metabolismo , Modelos Biológicos , Modelos Químicos , Piperidinas/farmacologia , Tamoxifeno/farmacologia , Ativação TranscricionalRESUMO
Rho GTPases are crucial regulators of the actin cytoskeleton and they play a role in the control of membrane trafficking. In contrast to the close family members RhoA and RhoC, RhoB localises to endosomes and delays epidermal growth factor receptor traffic. Here, we show that activated RhoB induces the peripheral distribution of endosomes, which align along subcortical actin stress fibres and are surrounded by an actin coat. The Diaphanous-related formin, Dia1, is recruited to endosomes by activated RhoB. Dia1 is required for the formation of the actin coat around endosomes downstream of RhoB, connecting membrane trafficking with the regulation of actin dynamics.
Assuntos
Actinas/química , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Proteína rhoB de Ligação ao GTP/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Forma Celular , Citoesqueleto/metabolismo , Endocitose , Forminas , Células HeLa , Humanos , Transdução de SinaisRESUMO
Using a novel approach that detects changes in the conformation of ERalpha, we studied the efficacy of anti-estrogens to inactivate ERalpha under different experimental conditions. We show that phosphorylation of serine-305 in the hinge region of ERalpha by protein kinase A (PKA) induced resistance to tamoxifen. Tamoxifen bound but then failed to induce the inactive conformation, invoking ERalpha-dependent transactivation instead. PKA activity thus induces a switch from antagonistic to agonistic effects of tamoxifen on ERalpha. In clinical samples, we found that downregulation of a negative regulator of PKA, PKA-RIalpha, was associated with tamoxifen resistance prior to treatment. Activation of PKA by downregulation of PKA-RIalpha converts tamoxifen from an ERalpha inhibitor into a growth stimulator, without any effect on ICI 182780 (Fulvestrant).
Assuntos
Neoplasias da Mama/patologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Resistencia a Medicamentos Antineoplásicos , Estradiol/análogos & derivados , Receptores de Estrogênio/química , Tamoxifeno/farmacologia , Antineoplásicos Hormonais/farmacologia , Sítios de Ligação , Western Blotting , Neoplasias da Mama/enzimologia , Divisão Celular , Linhagem Celular Tumoral , Regulação para Baixo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Estradiol/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio , Feminino , Transferência Ressonante de Energia de Fluorescência , Fulvestranto , Humanos , Luciferases/metabolismo , Microscopia Confocal , Modelos Biológicos , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Conformação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Receptores de Estrogênio/metabolismo , Serina/química , Fatores de Tempo , Ativação Transcricional , TransfecçãoRESUMO
Estrogen receptor-mediated transcription is enhanced by overexpression of G1/S cyclins D1, E or A in the presence as well in the absence of estradiol. Excess of G1/S cyclins also prevents the inhibition of transactivation of estrogen receptor (ER) by the pure antiestrogen ICI 182780. Cyclin D1 mediates this transactivation independent of complex formation to its CDK4/6 partner. This raises the possibility that overexpression of G1/S cyclins renders growth of ER-positive breast cancer hormone-independent and resistant to treatment with antiestrogens. Transient transfection of ER-positive breast cancer cell lines T47D and MCF7 with G1/S cyclins could overcome the growth arrest induced by ICI 182780 treatment. The ability of various cyclin D1 mutants to overcome the ICI 182780 mediated growth arrest corresponded with their ability to stimulate cyclin A- and E2F- promoter based reporter activities in the presence of ICI 182780. Transfection of a mutant cyclin D1 (cyclin D1-KE) that was unable to bind CDK4 and was reported to transactivate ER in the presence of ICI 182780, could not stimulate proliferation in ICI 182780 treated cells. On the other hand, cyclin D1-LALA, which is unable to stimulate ERE transactivation, could overcome the ICI 182780 cell cycle arrest. Furthermore, transient transfection of T47D cells using cyclin D1 together with a catalytic inactive mutant of CDK4 (CDK4-DN) indicated that the observed effect is due to binding to CDK inhibitors. However, a moderate, sixfold overexpression of cyclin D1 in stably transfected MCF7 cells did not overcome the ICI 182780 mediated growth arrest. These results indicate that CDK-independent transactivation of the estrogen receptor by cyclin D1 is by itself, not sufficient to result in estradiol-independent growth of breast cancer cells, whereas a vast overexpression of G1/S cyclins is able to do so, most likely by capturing of CDK inhibitors.