RESUMEN
The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.
Asunto(s)
Bioquímica , Biología Molecular , Publicaciones Periódicas como Asunto/estadística & datos numéricos , Edición/tendencias , Investigación , Brasil , Humanos , Publicaciones Periódicas como Asunto/normas , Publicaciones Periódicas como Asunto/tendenciasRESUMEN
The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.
Asunto(s)
Humanos , Publicaciones Periódicas como Asunto/estadística & datos numéricos , Edición/tendencias , Investigación , Bioquímica , Biología Molecular , Publicaciones Periódicas como Asunto/normas , Publicaciones Periódicas como Asunto/tendencias , BrasilRESUMEN
Intratumoral heterogeneity (ITH) represents an obstacle for cancer diagnosis and treatment, but little is known about its functional role in cancer progression. The A Desintegrin And Metalloproteinase 23 (ADAM23) gene is epigenetically silenced in different types of tumors, and silencing is often associated with advanced disease and metastasis. Here, we show that invasive breast tumors exhibit significant ADAM23-ITH and that this heterogeneity is critical for tumor growth and metastasis. We demonstrate that while loss of ADAM23 expression enhances invasion, it causes a severe proliferative deficiency and is not itself sufficient to trigger metastasis. Rather, we observed that, in ADAM23-heterotypic environments, ADAM23-negative cells promote tumor growth and metastasis by enhancing the proliferation and invasion of adjacent A23-positive cells through the production of LGI4 (Leucine-rich Glioma Inactivated 4) and nitric oxide (NO). Ablation of LGI4 and NO in A23-negative cells significantly attenuates A23-positive cell proliferation and invasion. Our work denotes a driving role of ADAM23-ITH during disease progression, shifting the malignant phenotype from the cellular to the tissue level. Our findings also provide insights for therapeutic intervention, enforcing the need to ascertain ITH to improve cancer diagnosis and therapy.
Asunto(s)
Proteínas ADAM/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de la Matriz Extracelular/metabolismo , Óxido Nítrico/metabolismo , Proteínas ADAM/genética , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular , Metilación de ADN , Epigénesis Genética , Proteínas de la Matriz Extracelular/genética , Femenino , Silenciador del Gen , Humanos , Metástasis de la Neoplasia , Proteínas del Tejido Nervioso , Carga Tumoral , Microambiente TumoralRESUMEN
Serum amyloid A (SAA) levels are elevated highly in acute phase response and elevated slightly and persistently in chronic diseases such as rheumatoid arthritis and diabetes. Given that fibroblasts exert profound effects on progression of inflammatory chronic diseases, the aim of this study was to investigate the response of fibroblasts to SAA. A dose-dependent increase in O(2) (-) levels was observed by treatment of fibroblasts with SAA (r = 0·99 and P ≤ 0·001). In addition, the expression of p47-phox was up-regulated by SAA (P < 0·001) and diphenyliodonium (DPI), a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, reduced the release of O(2) (-) by 50%. Also, SAA raised fibroblast proliferation (P < 0·001) and this effect was completely abolished by the addition of anti-oxidants (P < 0·001). These findings support the notion that, in chronic inflammatory sites, SAA activated fibroblast proliferation and ROS production.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína Amiloide A Sérica/farmacología , Animales , Antioxidantes/farmacología , Compuestos de Bifenilo/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Ratones , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , Compuestos Onio/farmacología , Células 3T3 SwissRESUMEN
In G(0)/G(1) cell cycle-arrested mouse Y1 adrenocortical cells, short pulses (30 min to 2 h) of fibroblast growth factor-2 (FGF2) (5 pM to 1 nM) caused induction of cFos protein by 2 h and onset of DNA synthesis stimulation by 8-9 h. FGF2 dose-response curves for cFos induction (percent labeled nuclei with a specific anti-cFos antibody) and DNA synthesis stimulation (bromodeoxyuridine labeling index) were linearly correlated with a correlation coefficient of 0.969. Inhibition of cFos and cJun protein induction with antisense oligodeoxynucleotides (ODNs) to cfos and cjun mRNAs blocked DNA synthesis stimulation by FGF2. Pulses (up to 2 h) of synthetic ACTH(39) (1 pM to 1 nM) and natural porcine corticotropin A (10 pg/ml to 1 microg/ml) also induced cFos protein and DNA synthesis in G(0)/G(1)-arrested Y1 adrenal cells. ACTH dose-response curves for cFos induction and DNA synthesis stimulation were not correlated. But cfos and/or cjun antisense ODNs blocked DNA synthesis stimulation by ACTH. Thus, signals initiated in FGF2 and ACTH receptors appear to converge to the induction of cfos and cjun genes to trigger DNA synthesis stimulation.
Asunto(s)
Hormona Adrenocorticotrópica/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Mitosis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/fisiología , Proteínas Proto-Oncogénicas c-jun/fisiología , Corteza Suprarrenal/citología , Animales , Relación Dosis-Respuesta a Droga , Ratones , Oligonucleótidos Antisentido/genética , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/genética , ARN Mensajero/genética , Fase S/genética , Células Tumorales CultivadasRESUMEN
This article reviews recent results of studies aiming to elucidate modes of integrating signals initiated in ACTH receptors and FGF2 receptors, within the network system of signal transduction found in Y1 adrenocortical cells. These modes of signal integration should be central to the mechanisms underlying the regulation of the G0->G1->S transition in the adrenal cell cycle. FGF2 elicits a strong mitogenic response in G0/G1-arrested Y1 adrenocortical cells, that includes a) rapid and transient activation of extracellular signal-regulated kinases-mitogen-activated protein kinases (ERK-MAPK) (2 to 10 min), b) transcription activation of c-fos, c-jun and c-myc genes (10 to 30 min), c) induction of c-Fos and c-Myc proteins by 1 h and cyclin D1 protein by 5 h, and d) onset of DNA synthesis stimulation within 8 h. ACTH, itself a weak mitogen, interacts with FGF2 in a complex manner, blocking the FGF2 mitogenic response during the early and middle G1 phase, keeping ERK-MAPK activation and c-Fos and cyclin D1 induction at maximal levels, but post-transcriptionally inhibiting c-Myc expression. c-Fos and c-Jun proteins are mediators in both the strong and the weak mitogenic responses respectively triggered by FGF2 and ACTH. Induction of c-Fos and stimulation of DNA synthesis by ACTH are independent of PKA and are inhibited by the PKC inhibitor GF109203X. In addition, ACTH is a poor activator of ERK-MAPK, but c-Fos induction and DNA synthesis stimulation by ACTH are strongly inhibited by the inhibitor of MEK1 PD98059.
Asunto(s)
Humanos , Animales , Corteza Suprarrenal/citología , Receptores de Corticotropina/fisiología , Transducción de Señal/fisiología , Neoplasias de la Corteza Suprarrenal , Ciclo Celular/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Células Tumorales Cultivadas/fisiologíaRESUMEN
This article reviews recent results of studies aiming to elucidate modes of integrating signals initiated in ACTH receptors and FGF2 receptors, within the network system of signal transduction found in Y1 adrenocortical cells. These modes of signal integration should be central to the mechanisms underlying the regulation of the G0-->G1-->S transition in the adrenal cell cycle. FGF2 elicits a strong mitogenic response in G0/G1-arrested Y1 adrenocortical cells, that includes a) rapid and transient activation of extracellular signal-regulated kinases-mitogen-activated protein kinases (ERK-MAPK) (2 to 10 min), b) transcription activation of c-fos, c-jun and c-myc genes (10 to 30 min), c) induction of c-Fos and c-Myc proteins by 1 h and cyclin D1 protein by 5 h, and d) onset of DNA synthesis stimulation within 8 h. ACTH, itself a weak mitogen, interacts with FGF2 in a complex manner, blocking the FGF2 mitogenic response during the early and middle G1 phase, keeping ERK-MAPK activation and c-Fos and cyclin D1 induction at maximal levels, but post-transcriptionally inhibiting c-Myc expression. c-Fos and c-Jun proteins are mediators in both the strong and the weak mitogenic responses respectively triggered by FGF2 and ACTH. Induction of c-Fos and stimulation of DNA synthesis by ACTH are independent of PKA and are inhibited by the PKC inhibitor GF109203X. In addition, ACTH is a poor activator of ERK-MAPK, but c-Fos induction and DNA synthesis stimulation by ACTH are strongly inhibited by the inhibitor of MEK1 PD98059.
Asunto(s)
Corteza Suprarrenal/citología , División Celular/fisiología , Receptores de Corticotropina/fisiología , Transducción de Señal/fisiología , Neoplasias de la Corteza Suprarrenal , Animales , Ciclo Celular/fisiología , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Células Tumorales Cultivadas/fisiologíaRESUMEN
In G0/G1 cell cycle arrested mouse Y1 adrenocortical tumor cells ACTH39, a weak mitogen and strong anti-mitogenic agent, blocks FGF2 mitogenic activity at G1 phase, keeping untouched ERK-MAPK activation and c-Fos protein induction. Here we report two anti-mitogenic mechanisms initiated in ACTH receptors and mediated by cAMP/PKA: a) post-transcriptional down regulation of c-Myc protein; b) dephosphorylation of AKT/PKB. In Y-1 cells the activity of the Mad/Max/Myc network of transcription factors seems to be regulated by c-Myc levels. FGF2 induces c-myc gene and stabilizes c-Myc protein by a process dependent on ERK-MAPK (PD98059 sensitive), but not on PI3K (Wortmannin resistant). ACTH39, on the other hand, causes rapid decrease in c-Myc levels induced by FGF2 in wild type Y1 cells, but not in PKA-deficient Y1 clones. The ACTH inhibition of DNA synthesis stimulated by FGF2 is reversed by transient transfection and induction of the MycER chimera (fusion of c-Myc and estrogen-receptor), suggesting that c-Myc down regulation is an efficient anti-mitogenic mechanism activated by ACTH. Y1 cells display high constitutive levels of AKT/PKB, that is dependent on elevated Ras x GTP. FGF2 up regulates Ras x GTP, PI3K and AKT/PKB. ACTH antagonizes this mitogenic effect of FGF2, promoting rapid dephosphorylation of AKT/PKB.
Asunto(s)
Corteza Suprarrenal/patología , Hormona Adrenocorticotrópica/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fase G1/fisiología , Proteínas Serina-Treonina Quinasas , Fase de Descanso del Ciclo Celular/fisiología , Transducción de Señal/efectos de los fármacos , Corteza Suprarrenal/efectos de los fármacos , Animales , Regulación hacia Abajo , Ratones , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-myc/metabolismo , Células Tumorales CultivadasRESUMEN
In G0/G1 cell cycle-arrested Y1 adrenocortical cells FGF2 is a strong mitogen, whereas ACTH39 can be a weak mitogen or a strong anti-mitogenic agent. Phosphorylated ERK1/2-MAP kinases are undetectable by Western and immunocitochemistry assay in G0/G1-arrested Y1 adrenal cells. Cell entry into S phase linearly correlates with migration of phosphorylated ERK to nucleus. FGF2 rapid and strongly triggers transient phosphorylation of ERK1/2, whereas ACTH39 is a poor ERK1/2 activator. But, the MEK1 inhibitor, PD98059 (50microM), inhibits cFos and cyclin D1 induction and DNA synthesis stimulation by both ACTH39 and FGF2, suggesting that ERK1/2 activation mediates the strong and the weak mitogenic effect of, respectively, FGF2 and ACTH39. In addition, ACTH39 antagonizes the FGF2 mitogenic effect keeping untouched ERK1/2 activation, c-Fos and cyclin D1 induction.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/patología , Hormona Adrenocorticotrópica/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Mitógenos/farmacología , Animales , Ciclina D1/metabolismo , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-fos/metabolismoRESUMEN
Mouse Y1 adrenocortical tumor cells harbor amplified and overexpressed c-Ki-ras gene, displaying relatively high constitutive levels of Ras x GTP. Here we report that Y1 cells also constitutively display high levels of phosphorylated AKT/PKB, that are dependent on Ras x GTP and PI3K. ACTH rapidly causes dephosphorylation of AKT/PKB in a cAMP/PKA dependent maner. This ACTH inhibition of the anti-apoptic and mitogenic AKT/PKB pathway is likely to be relevant in ACTH growth inhibitory effects in Y-adrenocortical cells.
Asunto(s)
Corteza Suprarrenal/fisiología , Hormona Adrenocorticotrópica/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Mitógenos/fisiología , Proteínas Serina-Treonina Quinasas , Sulfonamidas , Proteínas ras/fisiología , Corteza Suprarrenal/citología , Androstadienos/farmacología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Dexametasona/farmacología , Inhibidores Enzimáticos/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Glucocorticoides/farmacología , Isoquinolinas/farmacología , Ratones , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Células Tumorales Cultivadas , Wortmanina , Proteínas ras/antagonistas & inhibidoresRESUMEN
FGF2 elicits a strong mitogenic response in the mouse Y-1 adrenocortical tumor cell line, that includes a rapid and transient activation of the ERK-MAPK cascade and induction of the c-Fos protein. ACTH, itself a very weak mitogen, blocks the mitogenic response effect of FGF2 in the early and middle G1 phase, keeping both ERK-MAPK activation and c-Fos induction at maximal levels. Probing the mitogenic response of Y-1 cells to FGF2 with ACTH is likely to uncover reactions underlying the effects of this hormone on adrenocortical cell growth.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/fisiopatología , Hormona Adrenocorticotrópica/fisiología , Factores de Crecimiento de Fibroblastos/fisiología , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteínas Proto-Oncogénicas c-fos/fisiología , Animales , Transformación Celular Neoplásica/efectos de los fármacos , RatonesRESUMEN
FGF2 elicits a strong mitogenic response in the mouse Y-1 adrenocortical tumor cell line, that includes a rapid and transient activation of the ERK-MAPK cascade and induction of the c-Fos protein. ACTH, itself a very weak mitogen, blocks the mitogenic response effect of FGF2 in the early and middle G1 phase, keeping both ERK-MAPK activation and c-Fos induction at maximal levels. Probing the mitogenic response of Y-1 cells to FGF2 with ACTH is likely to uncover reactions underlying the effects of this hormone on adrenocortical cell growth
Asunto(s)
Animales , Ratones , Neoplasias de la Corteza Suprarrenal , Hormona Adrenocorticotrópica/farmacología , Proteína Quinasa 1 Activada por Mitógenos/farmacología , Proteínas Proto-Oncogénicas c-fos/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Interacciones FarmacológicasRESUMEN
Fetal calf serum (FCS) and PMA (phorbol 12-myristate-13-acetate) specifically stimulate the synthesis of heparan sulfate proteoglycan in endothelial cells. Staurosporine and n-butanol, kinase inhibitors, abolish the PMA effect. Forskolin and 8-bromo adenosine 3':5'-cyclic monophosphate, activators of, respectively, adenylate cyclase and protein kinase A cannot reproduce the PMA effect. The kinetics of cell entry into S phase of the endothelial cells was determined by DNA synthesis ([3H]-thymidine and Br-dU incorporation), and flow cytometry. The mitogenic effect of fetal calf serum is abolished by PMA. Also, PMA pre-treatment inhibits the enhanced synthesis of heparan sulfate proteoglycan after a second PMA exposure. Remarkably, the stimulation of heparan sulfate proteoglycan synthesis by fetal calf serum and PMA seems to be mainly restricted to G1 phase. Therefore fetal calf serum and PMA cause an enhanced synthesis of heparan sulfate proteoglycan, and PMA causes a cell cycle block at G1 phase.
Asunto(s)
Fase G1 , Sustancias de Crecimiento/farmacología , Proteoglicanos de Heparán Sulfato/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Animales , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Endotelio Vascular/metabolismo , Activación Enzimática , Citometría de Flujo , Proteoglicanos de Heparán Sulfato/metabolismo , Proteína Quinasa C/metabolismo , ConejosRESUMEN
The entire ACTH receptor (ACTH-R) cDNA was amplified by RT/PCR from mouse Y-1 adrenocortical cells, subcloned into the pMOSBlue T vector, sequenced and inserted into the pSVK3 mammalian vector to obtain pSVACTHR. Balb 3T3 fibroblasts were co-transfected with pSVACTHR plus pSV2-neo and the transfectants were selected with G418 and cloned. Genomic integration of pSVACTHR and transcription of ACTH-R cDNA were checked by Southern blot and RT/PCR respectively. Expression of active ACTH-R protein was tested by measuring cAMP production in response to ACTH. Two ACTH-R expressing transfectants (clones 03 and 07) increased cAMP accumulation in response to ACTH. They were morphologically identical to parental 3T3 cells, but required 10-20% FCS to grow. In these transfectants, ACTH induced c-FOS protein expression, but did not activate the ERK isoforms of MAP Kinase and did not stimulate DNA synthesis. Apparently, the ACTH-R in Balb 3T3 cells induces the c-fos gene by a pathway independent of cAMP/protein kinase A and ERK/MAP Kinase.
Asunto(s)
Hormona Adrenocorticotrópica/farmacología , Fibroblastos/fisiología , Regulación de la Expresión Génica/fisiología , Proteínas Proto-Oncogénicas c-fos/genética , Proto-Oncogenes/genética , Receptores de Corticotropina/metabolismo , Células 3T3 , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adenilil Ciclasas/metabolismo , Animales , Bucladesina/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , ADN/biosíntesis , Fibroblastos/metabolismo , Ratones , Fosforilación/efectos de los fármacos , Transfección/fisiologíaRESUMEN
Pulses (up to 2 h) of the adrenocorticotropic hormone (ACTH) rapidly activate p42 and p44 MAPK (5 min), induce the c-Fos protein (1 h, 80% of cells) and stimulate entry of mouse Y-1 adrenocortical cells into the S phase of the cell cycle. This set of sequential events is also triggered in Y-1 cells by bFGF, and reflects a mitogenic response to ACTH. We report here that 90% inhibition of c-fos mRNA translation with a c-fos antisense oligodeoxynucleotide completely blocks the entry of Y1 cells into S phase stimulated by pulses of ACTH. These results indicate that c-Fos protein is an intracellular mediator of the mitogenic response to ACTH.
Asunto(s)
Hormona Adrenocorticotrópica/farmacología , Mitógenos/farmacología , Proteínas Proto-Oncogénicas c-fos/fisiología , Neoplasias de la Corteza Suprarrenal/genética , Neoplasias de la Corteza Suprarrenal/metabolismo , Neoplasias de la Corteza Suprarrenal/patología , Hormona Adrenocorticotrópica/antagonistas & inhibidores , Animales , ADN/biosíntesis , Factores de Crecimiento de Fibroblastos/farmacología , Ratones , Mitógenos/antagonistas & inhibidores , Oligonucleótidos Antisentido/farmacología , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/genética , Fase S/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patologíaRESUMEN
The adrenocorticotropic hormone (ACTH) inhibits the growth of Y1 mouse adrenocortical tumor cells as well as normal adrenocortical cells in culture but stimulates adrenocortical cell growth in vivo. In this study, we investigated this paradoxical effect of ACTH on cell proliferation in Y1 adrenal cells and have unmasked a growth-promoting effect of the hormone. Y1 cells were arrested in the G1 phase of the cell cycle by serum starvation and monitored for progression through S phase by measuring [3H]thymidine incorporation into DNA and by measuring the number of nuclei labeled with bromodeoxyuridine. Y1 cells were stimulated to progress through S phase and to divide after a brief pulse of ACTH (up to 2 h). This effect of ACTH appeared to be cAMP independent, since ACTH also induced cell cycle progression in Kin-8, a Y1 mutant with defective cAMP-dependent protein kinase activity. The growth-promoting effect of ACTH in Y1 was preceded by the rapid activation of p44 and p42 mitogen-activated protein kinases and by the accumulation of c-FOS protein. In contrast, continuous treatment with ACTH (14 h) inhibited cell cycle progression in Y1 cells by a cAMP-dependent pathway. The inhibitory effect of ACTH mapped to the midpoint of G1. Together, the results demonstrate a dual effect of ACTH on cell cycle progress, a cAMP-independent growth-promoting effect early in G1 possibly mediated by mitogen-activated protein kinase and c-FOS, and a cAMP-dependent inhibitory effect at mid-G1. It is suggested that the growth-inhibitory effect of ACTH at mid-G1 represents an ACTH-regulated check point that limits cell cycle progression.
Asunto(s)
Corteza Suprarrenal/citología , Hormona Adrenocorticotrópica/fisiología , Ciclo Celular/fisiología , Sustancias de Crecimiento/fisiología , Proteínas Quinasas Activadas por Mitógenos , Neoplasias de la Corteza Suprarrenal , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , División Celular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Fase G1 , Ratones , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Fosforilación , Proteínas Proto-Oncogénicas c-fos/metabolismo , Fase S , Células Tumorales CultivadasRESUMEN
Y-1 adrenal cells were cell cycle arrested by serum starvation to characterize a G0-->G1-->S transition in these cells. Cycle arrested Y-1 cells start to enter S phase 8h after serum feeding, reaching more than 90% cells synthesizing DNA by 24h. ACTH displays a dual effect in the G0-->G1-->S transition: 2h ACTH treatment stimulates DNA synthesis initiation, but longer treatments inhibit S phase entry. This dual effect of ACTH is similar to the antagonistic actions of PMA (phorbol-12-miristate-13-acetate) on the G0-->G1-->S transition. However ACTH and PMA are likely to have different mechanisms of action. ACTH inhibitory effect requires PKA, whereas PMA inhibitory effect is not dependent on PKA. ACTH induces the proto-oncogenes c-fos and c-jun, but inhibits the expression of the c-myc proto-oncogene. PMA, on the other hand, induces equally well c-fos, c-jun and c-myc. We hypothesize that ACTH promotes G0-->G1 transition by induction of c-fos and c-jun and blocks G1-->S transition by c-myc inhibition.
Asunto(s)
Corteza Suprarrenal/citología , Hormona Adrenocorticotrópica/farmacología , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células 3T3/citología , Neoplasias de la Corteza Suprarrenal , Animales , ADN/biosíntesis , Fase G1/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Genes fos/genética , Genes jun/genética , Genes myc/genética , Cinética , Ratones , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Fase S/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
Adrenocorticotropic hormone (ACTH) triggers well-defined responses in Y-1 cells. Among them is steroidogenesis stimulation. We have previously shown that phorbol 12-myristate 13-acetate (PMA), an activator of the calcium- and phospholipid-dependent protein kinase (PKC) is able to mimic all the responses triggered by ACTH in these cells, including steroidogenesis stimulation. Short (2 h) treatment with PMA leads to only 20-30% of the maximal steroidogenesis stimulation obtained with ACTH. However, the steroid secretion in the 2 h that follows the short-term (2 h) PMA treatment reaches the same levels as observed with ACTH, i.e., a 12- to 15-fold increase. We also show that this effect is restricted to cells treated with PMA for up to 4 h, while treatment for longer periods of time causes a reduction of the steroid biosynthesis rate, an effect that is not observed in cells treated with ACTH or N6,2'-0-dibutyryladenosine 3',5'-cyclic monophosphate (dcAMP). These results suggest that activation of PKC can elicit the first phase of ACTH steroidogenesis stimulation, but not the second one, which strictly depends on activation of cAMP-dependent protein kinase.
Asunto(s)
Hormona Adrenocorticotrópica/metabolismo , AMP Cíclico/metabolismo , Ésteres del Forbol/metabolismo , Esteroides/biosíntesis , Hormona Adrenocorticotrópica/fisiologíaRESUMEN
Adrenocorticotropic hormone (ACTH) triggers well-defined responses in Y-1 cells. Among them is steroidogenesis stimulation. We have previously shown that phorbol 12-myristate 13-acetate (PMA), an activator of the calcium- and phospholipid-dependent protein kinase (PKC) is able to mimic all the responses triggered by ACTH in these cells, including steroidogenesis stimulation. Short (2 h) treatment with PMA leads to only 20-30 per cent of the maximal steroidogenesis stimulation obtained with ACTH. However, the steroid secretion in the 2 h that follows the short-term (2 h) PMA treatment reaches the same levels as observed with ACTH, i.e., a 12- to 15-fold increase. We also show that this effect is restricted to cells treated with PMA for up to 4 h, while treatment for longer periods of time causes a reduction of the steroid biosynthesis rate, an effect that is not observed in cells treated with ACTH or N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (dcAMP). These results suggest that activation of PKC can elicit the first phase of ACTH steroidogenesis stimulation, but not the second one, which strictly depends on activation of cAMP-dependent protein kinase.
Asunto(s)
Hormona Adrenocorticotrópica/metabolismo , AMP Cíclico/metabolismo , Ésteres del Forbol/metabolismo , Esteroides/biosíntesis , Hormona Adrenocorticotrópica/fisiologíaRESUMEN
We have developed a protocol that reveals two antagonistic effects of phorbol-12-myristate-12-acetate (PMA) on the G0-->G1-->S transition of mammalian cell cycle. Balb-3T3 (Clone A31) cells arrested in G0 by serum starvation can be stimulated to traverse the G1 phase and initiate DNA synthesis 12 h later by a 2-h pulse with PMA. In contrast with this early stimulatory effect, PMA has an inhibitory effect when presented to the cells during the last 6 h of G1. PMA is able to inhibit DNA synthesis initiation irrespective of the triggering agent, i.e., serum, fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, or PMA itself (presented as an early pulse). We have established that the critical period for the PMA inhibitory effect is between 6 and 8 h after cell stimulation. This dual effect of PMA is not a peculiarity of Balb-3T3 (clone A31) cells because it is also observed with other fibroblastic cell lines, namely, SWISS 3T3, NIL 8, and RAT 1, and also with the epithelial Y-1 adrenocortical cell line. Treatment with PMA for 0.5 or 2 h activates protein kinase C (PKC) in Balb-3T3-A31 cells, but is not sufficient to down-regulate the enzyme because a second 30-min PMA pulse applied between 6 and 6.5 h activates PKC again. On the other hand, a continuous 6.5-h PMA treatment causes PKC down-regulation; therefore, the inhibitory effect of PMA could be mediated by PKC. Growth factor early response proto-oncogenes c-myc, c-fos, and c-jun are induced transiently by both early and late PMA pulses, suggesting that these genes are not involved in the PMA inhibitory effect.