RESUMEN

The activation of the growth arrest-specific (gas) p20K gene depends on the interaction of C/EBPß with two elements of a 48-bp promoter region termed the quiescence-responsive unit (QRU). Here we identify extracellular signal-related kinase 2 (ERK2) as a transcriptional repressor of the p20K QRU in cycling chicken embryo fibroblasts (CEF). ERK2 binds to repeated GAAAG sequences overlapping the C/EBPß sites of the QRU. The recruitment of ERK2 and C/EBPß is mutually exclusive and dictates the expression of p20K. C/EBP homologous protein (CHOP) was associated with C/EBPß under conditions promoting endoplasmic reticulum (ER) stress and, to a lesser extent, in cycling CEF but was not detectable when C/EBPß was immunoprecipitated from contact-inhibited cells. During ER stress, overexpression of CHOP inhibited p20K, while its downregulation promoted p20K, indicating that CHOP is also a potent inhibitor of p20K. Transcriptome analyses revealed that hypoxia-responsive genes are strongly induced in contact-inhibited but not serum-starved CEF, and elevated levels of nitroreductase activity, a marker of hypoxia, were detected at confluence. Conditions of hypoxia (2% O2) induced growth arrest in subconfluent CEF and markedly stimulated p20K expression, suggesting that the control of proliferation and gas gene expression is closely linked to limiting oxygen concentrations associated with high cell densities.

Asunto(s)

Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Fibroblastos/citología , Lipocalinas/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Factor de Transcripción CHOP/metabolismo , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Ciclo Celular , Hipoxia de la Célula , Proliferación Celular , Células Cultivadas , Embrión de Pollo , Estrés del Retículo Endoplásmico , Fibroblastos/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Regiones Promotoras Genéticas

RESUMEN

Triple-negative breast cancers (TNBCs) represent a subset of breast tumors that are highly aggressive and metastatic, and are responsible for a disproportionate number of breast cancer-related deaths. Several studies have postulated a role for the epithelial-to-mesenchymal transition (EMT) program in the increased aggressiveness and metastatic propensity of TNBCs. Although EMT is essential for early vertebrate development and wound healing, it is frequently co-opted by cancer cells during tumorigenesis. One prominent signaling pathway involved in EMT is the transforming growth factor-ß (TGFß) pathway. In this study, we report that the novel POZ-ZF transcription factor Kaiso is highly expressed in TNBCs and correlates with a shorter metastasis-free survival. Notably, Kaiso expression is induced by the TGFß pathway and silencing Kaiso expression in the highly invasive breast cancer cell lines, MDA-MB-231 (hereafter MDA-231) and Hs578T, attenuated the expression of several EMT-associated proteins (Vimentin, Slug and ZEB1), abrogated TGFß signaling and TGFß-dependent EMT. Moreover, Kaiso depletion attenuated the metastasis of TNBC cells (MDA-231 and Hs578T) in a mouse model. Although high Kaiso and high TGFßR1 expression is associated with poor overall survival in breast cancer patients, overexpression of a kinase-active TGFßR1 in the Kaiso-depleted cells was insufficient to restore the metastatic potential of these cells, suggesting that Kaiso is a key downstream component of TGFß-mediated pro-metastatic responses. Collectively, these findings suggest a critical role for Kaiso in TGFß signaling and the metastasis of TNBCs.

RESUMEN

Nutrient depletion triggers a series of adaptive processes as part of the unfolded protein response or UPR. These processes reduce stress to the endoplasmic reticulum by enhancing its protein folding capacity or ability to promote the degradation of dysfunctional proteins. Failure to restore ER homeostasis causes the activation of lethal pathways. The expression of a dominant negative mutant of C/EBPß (Δ184-C/EBPß) alters this balance in chicken embryo fibroblasts (CEF). As a result, CEF display enhanced survival upon prolonged nutrient depletion. Starved Δ184-C/EBPß-expressing CEF display pronounced features of autophagy characterized by the appearance of large vesicles containing amorphous material, the formation of smaller double-membrane vesicles (autophagosomes) and processing of LC3 and GABARAP. However, there were marked differences in the expression and processing of these proteins. In both normal and Δ184-C/EBPß expressing CEF, the lipidated form of LC3 (form II) accumulated during starvation but was detectable even when cells were actively dividing in complete medium. In contrast, GABARAP expression and lipidation were strongly stimulated in response to starvation. Inhibition of LC3 expression by RNA interference led to apoptosis in normal CEF even in the absence of starvation but stable and near complete repression of GABARAP was tolerated. Moreover, the inhibition of GABARAP enhanced CEF survival and abolished the expression of the pro-apoptotic CHOP factor in conditions of starvation, suggesting a reduced level of ER stress. Therefore, GABARAP is a determinant of apoptosis in CEF subjected to prolonged nutrient depletion.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/fisiología , Fibroblastos/citología , Fibroblastos/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Técnicas de Cultivo de Célula , Proliferación Celular , Embrión de Pollo , Pollos , Regulación de la Expresión Génica/fisiología , Proteínas Asociadas a Microtúbulos/genética , Mutación , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo

RESUMEN

The p38 member of the mitogen-activated protein kinase superfamily is engaged by phosphorylation in response to environmental stress signals, and may have either permissive or inhibitor roles upon cell proliferation. The cell cycle in the proliferative zone of the retina is tightly controlled and proceeds in synchrony with interkinetic migration of the neuroblast nuclei. We examined the association of p38 kinase activity with the cell cycle in the normal, non-stressed retina of the developing rat, maintained either in vivo or in vitro. Using immunohistochemistry, we show that mitotic profiles in the developing retina are highly enriched for phosphorylated p38. Blockade of p38 activity with the chemical inhibitor SB203580 for 4 h transiently arrested cells at the metaphase-anaphase transition and induced cell death after 20 h. p38 inhibition induced an aberrant mitotic profile, with chromosomes arranged in one side of the cell. The data show that p38 is active during normal mitosis and we suggest that p38 is required for the proper cell cycle progression during metaphase-anaphase transition in retinal neuroblasts.

Asunto(s)

Envejecimiento/metabolismo , Animales Recién Nacidos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mitosis/fisiología , Retina/enzimología , Retina/crecimiento & desarrollo , Anafase/fisiología , Animales , Animales Recién Nacidos/genética , Animales Recién Nacidos/crecimiento & desarrollo , Ciclo Celular/efectos de los fármacos , Cromosomas/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Metafase/fisiología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Piridinas/farmacología , Ratas , Ratas Endogámicas , Ratas Long-Evans , Valores de Referencia , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

The CEF-4/9E3 chemokine gene is expressed constitutively in chicken embryo fibroblasts (CEF) transformed by the Rous sarcoma virus (RSV). This aberrant induction is controlled at the transcriptional and post-transcriptional levels. Transcriptional activation depends on multiple elements of the CEF-4 promoter composing a Src-responsive-Unit or SRU. The SRU includes a TPA responsive element, a PRDII/kappaB domain and a CAAT box. In this report, we identify C/EBPbeta as a component of the trans-acting factor interacting with the CAAT box of the CEF-4 promoter. In addition, we show that C/EBPbeta binds to a second element located in proximity of the TRE. A mutation of this distal CAAT box impaired the activation of the CEF-4 promoter by pp60(v-src) indicating that this element is also part of the SRU. Using the RCASBP retroviral vector, we expressed a dominant negative mutant of C/EBPbeta (designated Delta184-C/EBPbeta) in RSV-transformed CEF. Delta184-C/EBPbeta decreased the accumulation of the CEF-4 mRNA and activation of the CEF-4 promoter by pp60(v-src). The induction of the Cox-2 gene (CEF-147) was also reduced by Delta184-C/EBPbeta. The effect of the dominant negative mutant was observed within 1 h of the activation of a thermolabile pp60(v-src) suggesting that C/EBPbeta is an early target of v-src transformation. The dominant negative mutant did not inhibit the transformation of CEF by RSV and in fact accentuated the transformed cell phenotype. Therefore, the activation of C/EBPbeta is important for the expression of v-src regulated genes but is not required for the in vitro transformation of CEF by pp60(v-src).

Asunto(s)

Proteínas Aviares , Proteína beta Potenciadora de Unión a CCAAT/genética , Transformación Celular Viral/genética , Citocinas/genética , Genes src/genética , Animales , Virus del Sarcoma Aviar/genética , Sitios de Unión , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Embrión de Pollo , Citocinas/metabolismo , Fibroblastos/fisiología , Fibroblastos/virología , Regulación de la Expresión Génica , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Elementos de Respuesta/genética

RESUMEN

The p20K gene is induced in conditions of reversible growth arrest in chicken embryo fibroblasts (CEF). This expression is dependent on transcriptional activation and on a region of the promoter designated the quiescence-responsive unit (QRU). In this report, we describe the regulatory elements of the QRU responsible for activation in resting cells and characterize the trans-acting proteins interacting with these elements. We show that the QRU consists of functionally distinct domains including quiescence-specific and weak proliferation-responsive elements. The quiescence responsiveness of the QRU was mapped to two C/EBP binding sites, and the activity of the p20K promoter and its QRU was inhibited by the expression of a dominant negative mutant of C/EBPbeta in nondividing cells. The activation of QRU in response to serum starvation and contact inhibition correlated with the presence of a growth arrest-specific complex in electrophoretic mobility shift assays. This complex was supershifted by antibody for C/EBPbeta. C/EBPbeta accumulated in conditions of contact inhibition as a result of transcriptional activation. Therefore, C/EBPbeta was itself regulated as a growth arrest-specific gene in CEF. Finally, we show that the expression of p20K is regulated by linoleic acid, an essential fatty acid binding to p20K. The addition of linoleic acid to contact-inhibited CEF markedly repressed the synthesis of p20K without inducing mitogenesis. The activity of the QRU was inhibited by linoleic acid or the peroxisome proliferator-activated receptor PPARgamma2 in transient expression assays. Therefore, we have identified C/EBPbeta as a key activator of a growth arrest-specific gene in CEF and implicated an essential fatty acid, linoleic acid, in regulation of the QRU and the p20K lipocalin gene.

Asunto(s)

Proteínas Sanguíneas/genética , Regulación de la Expresión Génica , Proteínas de Neurofilamentos/fisiología , Activación Transcripcional , Animales , Proteínas Aviares , Secuencia de Bases , Sitios de Unión , Proteínas Sanguíneas/biosíntesis , División Celular , Células Cultivadas , Embrión de Pollo , ADN Complementario/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Ácido Linoleico/fisiología , Lipocalinas , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiología , Activación Transcripcional/efectos de los fármacos

RESUMEN

The CEF-4/9E3 gene is expressed aberrantly in chicken embryo fibroblasts transformed by the Rous sarcoma virus. This aberrant expression is dependent on transcriptional activation and on the stabilization of the CEF-4 mRNA. The characterization of the CEF-4 promoter indicated that three distinct regulatory elements corresponding to an AP-1 binding site, a PRDII/ kappaB domain and a CAAT box are involved in the activation by pp60v-src. Several v-src responsive genes are controlled by AP-1 and members of the Ets family but few appear to be dependent on NF-kappaB. In this study we characterize the expression of genes regulated by NF-kappaB in normal and RSV-transformed CEF. Run-on transcription analysis indicated that pp60v-src induces the transcription of several genes controlled by NF-kappaB but at different levels. While the transcription of CEF-4 was strongly stimulated, that of NF-kappaB1, c-rel, p53 or IkappaB-alpha was activated more modestly by pp60v-src. In addition the CEF-4 mRNA was the only mRNA species to accumulate significantly in transformed CEF. The ectopic expression of RelA or Rel resulted in the stimulation of the transcription of several known targets of NF-kappaB. However, the mRNA for IkappaB-alpha was the only mRNA species to accumulate considerably in the RelA- or Rel-expressing cells. Hence for most kappaB-controlled genes, transcriptional activation was not sufficient to obtain a significant increase in mRNA expression. Likewise, RelA or Rel enhanced the transcription of the CEF-4 gene without a significant accumulation of the CEF-4 mRNA. However, transformation by v-src caused a massive accumulation of the CEF-4 mRNA but not of other mRNA species in the RelA- and Rel-expressing cells. Transient expression assays, run-on transcription and Northern blotting analyses indicated that the effect of pp60v-src on CEF-4 expression was mediated predominantly at the post-transcriptional level in these cells. Therefore transcriptional and post-transcriptional mechanisms determine the restricted pattern of activation of kappaB-controlled genes in RSV-transformed CEF.

Asunto(s)

Proteínas Aviares , Citocinas/genética , Regulación de la Expresión Génica , FN-kappa B/farmacología , Proteína Oncogénica pp60(v-src)/farmacología , Procesamiento Proteico-Postraduccional , Animales , Transformación Celular Viral , Pollos

RESUMEN

The CEF-4/9E3 cytokine gene is expressed aberrantly in chicken embryo fibroblasts (CEF) transformed by the Rous sarcoma virus. The expression of CEF-4 is dependent on both transcriptional and post-transcriptional mechanisms of regulation. The characterization of the promoter region indicated that three distinct regulatory elements corresponding to an AP-1 binding site (or TRE), a PRDII/kappaB domain, and a CAAT box are involved in the activation by pp60(v-)src. In this report we investigate the signaling pathways controlling the expression of the TRE and PRDII domain. The expression of a dominant negative mutant of p21(ras) reduced the activity of both elements. In contrast a similar mutant of c-Raf-1 affected modestly the activation dependent on the TRE but not PRDII. The stress-activated protein kinase (SAPK)/Jun N-terminal kinase (JNK) pathway was important for the activity of PRDII and the TRE but was not markedly stimulated by pp60(v-)src. The addition of calphostin C and the inhibition of protein kinase C (PKC) diminished the accumulation of the CEF-4 mRNA and reduced the activity of a TRE-controlled promoter. Likewise, the depletion of PKC by chronic treatment with phorbol esters inhibited the activation of the TRE. Rous sarcoma virus-transformed CEF treated with calphostin C were also flatter, did not display a high degree of criss-crossing, and appeared morphologically normal. Hence PKC was important for the activation of AP-1 and the morphological transformation of CEF. The constitutive expression of CEF-4 was correlated with transformation only when dependent on the TRE. This was not true for PRDII, which was the only element required for the constitutive activation to the CEF-4 promoter in nontransformed cells treated chronically with phorbol esters.

Asunto(s)

Proteínas Aviares , Citocinas/genética , Regulación de la Expresión Génica , Proteína Oncogénica pp60(v-src)/metabolismo , Transducción de Señal , Factores de Transcripción , Animales , Virus del Sarcoma Aviar , Northern Blotting , Transformación Celular Viral , Embrión de Pollo , Proteínas de Unión al ADN/metabolismo , Cinética , Naftalenos/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-raf , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Mensajero/metabolismo , Factores de Transcripción del Factor Regulador X , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-1/metabolismo

RESUMEN

Several genes are induced constitutively in cells transformed by the v-src oncoprotein. This induction is generally dependent on the activation of transcription factors binding to src-responsive elements of the promoter. In previous studies, we showed that the induction of the CEF-4/9E3 cytokine gene by pp60v-src is dependent on the PRDII/kappa B domain of the promoter (Dehbi et al., 1992). In this investigation, we describe the activation of the HIV-1 LTR by pp60v-src and show that a region of 30 bp containing the two NF-kappa B binding sites is critical for activation of the promoter. The induction was dependent on transformation since non-transforming forms of pp60v-src had little or no effect on the promoter. The expression of proviral DNA and the release of p24 antigen were also increased by v-src indicating that viral replication was stimulated in src-transformed cells. The effect of v-src on HIV-1 gene expression occurred in the presence or in the absence of the tat viral trans-activator, in fibroblasts and in Jurkat T lymphocytes. These results indicate that several promoters controlled by PRDII/kappa B may be activated constitutively in v-src transformed cells and suggest that oncogenic tyrosine kinases may play a role in the induction of viruses with a PRDII/kappa B-controlled promoter.

Asunto(s)

Regulación Viral de la Expresión Génica , VIH-1/genética , Proteína Oncogénica pp60(v-src)/fisiología , Animales , Secuencia de Bases , Embrión de Pollo , Duplicado del Terminal Largo de VIH , Datos de Secuencia Molecular , FN-kappa B/fisiología , Regiones Promotoras Genéticas , TATA Box

RESUMEN

We described the synthesis of a quiescence-specific p20K protein in quiescent chicken heart mesenchymal cells and contact-inhibited chicken embryo fibroblasts (Bédard, P.-A., Balk, S.D., Gunther, H.S., Morisi, A., and Erikson, R.L. (1987a) Mol. Cell. Biol. 7, 1450-1459). We now report that the expression of p20K is enhanced in cells rendered quiescent by other conditions of growth arrest such as serum starvation or treatment with hydroxyurea. Chicken embryo fibroblasts transformed by the Rous sarcoma virus also expressed p20K upon serum/medium depletion. In all conditions investigated, the synthesis of p20K was correlated with decreased DNA synthesis, indicating that growth arrest regulates the expression of p20K in fibroblasts. The abundance of p20K mRNAs was elevated in quiescent cells, and the p20K gene was more active in nuclear run-on transcription assays in conditions of growth arrest. The p20K gene was isolated, and the promoter region was analyzed in transient expression assays. Serum starvation increased the activity of the promoter, indicating that the expression of p20K is controlled at least in part at the transcriptional level. Deletion analysis revealed that a region of less than 217 base pairs (bp) is sufficient for quiescence-dependent activity of the promoter. Within this region, a segment of 48 bp was essential to basal and quiescence-induced activity of the promoter in dividing and nondividing cells, respectively. The 48-bp region enhanced the activity of a minimal heterologous promoter in quiescent cells but had no effect in conditions of proliferation, indicating that it functions as a quiescence-responsive unit (QRU). Therefore, the transcription of the p20K gene in quiescent fibroblasts is controlled by the one or several growth-regulated elements located in the 48-bp QRU of the promoter.

Asunto(s)

Proteínas Sanguíneas/genética , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , TATA Box , Animales , Proteínas Aviares , Secuencia de Bases , Proteínas Sanguíneas/biosíntesis , División Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Embrión de Pollo , Medios de Cultivo , Medio de Cultivo Libre de Suero , Replicación del ADN , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Genes fos , Biblioteca Genómica , Hidroxiurea/farmacología , Lipocalinas , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Plásmidos , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Factor de Crecimiento Transformador beta/genética

RESUMEN

In response to interleukin 1 or tumor necrosis factor, human synovial cells and fibroblasts expressed several genes encoding known chemotactic factors or related proteins. Transcripts for interleukin 8 (IL-8), gro/MGSA, pAT 464, IP-10, pAT 744 and Monocyte Chemotactic and Activating Factor (MCAF) accumulated rapidly in IL-1 and TNF-treated cells. The inhibition of protein synthesis led to the superinduction of IL-8 and gro/MGSA mRNAs in IL-1, but not in TNF-treated cells. Thus, IL-1 and TNF are likely to regulate the expression of these mRNAs by different mechanisms. Important cell-specific differences in mRNA accumulation characterized the expression of chemotactic factor genes. Moreover, only a subset of the same genes was activated in quiescent cells stimulated by serum. Therefore, genes encoding closely related proteins each had a distinct pattern of expression. continuous stimulation of fibroblasts and synovial cells with IL-1 resulted in high and prolonged expression of IL-8 and gro/MGSA mRNAs. These results extend the list of chemotactic factor genes expressed by mesenchymal cells in vitro and suggest a pivotal role for these cells in processes such as chronic inflammation.

Asunto(s)

Quimiocinas CXC , Factores Quimiotácticos/genética , Fibroblastos/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Interleucina-1/fisiología , Líquido Sinovial/citología , Factor de Necrosis Tumoral alfa/fisiología , Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Northern Blotting , Células Cultivadas , Quimiocina CXCL1 , Factores Quimiotácticos/biosíntesis , Factores Quimiotácticos/metabolismo , Regulación de la Expresión Génica , Sustancias de Crecimiento/biosíntesis , Sustancias de Crecimiento/genética , Humanos , Interleucina-8/biosíntesis , Interleucina-8/genética , Familia de Multigenes , Osteoartritis/genética , Osteoartritis/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética

RESUMEN

Several genes expressed in response to growth factors are also regulated aberrantly in oncogenically transformed cells. The constitutive expression of genes encoding extracellular proteases, transcription factors, and cytokines is often correlated with cell transformation. In several instances, the uncontrolled expression of these genes is the result of transcriptional activation. Therefore, much attention has been devoted to the study of promoter function in transformed cells. We now review the results of recent investigations on transformation-dependent gene expression. The activation of several transcription factors in oncogenically-transformed cells is described. Results regarding the regulation of promoters through PRD II/kappa B are presented for cells transformed by a variety of oncogenes. Finally, we discuss the significance of transcription factor activation in the process of cell transformation.

Asunto(s)

Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , Animales , Secuencia de Bases , Transformación Celular Neoplásica/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Oncogenes , Transducción de Señal/genética , Transcripción Genética/fisiología , Activación Transcripcional

RESUMEN

The CEF-4/9E3 gene is expressed constitutively in Rous sarcoma virus (RSV)-transformed cells. This expression is largely determined by an increase in transcription of the gene. In this report, we characterize the regulatory elements responsible for the transformation-dependent activation of CEF-4/9E3. Three sequences corresponding to AP-1, PRD II/kappa B, and TAACGCAATT are involved in the process and therefore define the src-responsive unit (SRU) of the CEF-4 promoter. In constructs containing a deletion of the SRU, multiple copies of AP-1 or PRD II/kappa B, but not TAACGCAATT, led to activation of the promoter. Thus, factors interacting with these elements are constitutively activated in RSV-transformed chicken embryo fibroblasts. In agreement with the results of transient expression assays, protein binding to AP-1, PRD II/kappa B, and TAACGCAATT were more abundant in the nuclei of transformed cells. The expression of the CEF-4 promoter was investigated in cells infected by a temperature-sensitive mutant of RSV. No significant increase in CEF-4 promoter activity was detected early after activation of pp60v-src. In contrast, a substantial activation of the CEF-4 promoter was detected late after a temperature shift. Factors interacting with the TAACGCAATT, PRD II/kappa B, and AP-1 elements accumulated gradually over a period of several hours. Therefore, transcriptional activation plays an important role in the late, constitutive expression of the CEF-4 gene in stably transformed cells.

Asunto(s)

Proteínas Aviares , Citocinas/genética , Regulación de la Expresión Génica , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transcripción Genética , Animales , Secuencia de Bases , Transformación Celular Viral , Células Cultivadas , Embrión de Pollo , Proteínas de Unión al ADN , Fibroblastos , Genes src , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Transformación Genética

RESUMEN

The transformation of chicken embryo fibroblasts (CEF) by the Rous sarcoma virus leads to the constitutive expression of the cellular gene designated CEF-4. With specific antisera, we confirmed that transformed cells actively synthesize and secrete a 6 kDa polypeptide corresponding to the CEF-4 gene product. The expression of CEF-4 was investigated by Northern and immunoprecipitation analyses. Upon activation of pp60v-src in cells infected by a ts mutant of RSV, the expression of CEF-4 was biphasic with an early transient and a late constitutive period of expression. CEF-4 was expressed in cells transformed by a variety of oncogenes, but the level of constitutive expression differed quantitatively among transformed cells. Cells transformed by v-myc alone did not express CEF-4. Unlike other members of the interleukin 8 gene family, CEF-4 was induced in response to a broad spectrum of growth factors and inflammatory agents. Dexamethasone repressed the induction of CEF-4 by lipopolysaccharides but had little or no effect on the response to serum or pp60v-src. These data emphasize the complexity of CEF-4 expression and suggest the existence of multiple levels or pathways of CEF-4 regulation in normal and transformed cells.

Asunto(s)

Proteínas Aviares , Virus del Sarcoma Aviar/genética , Transformación Celular Viral/genética , Citocinas/genética , Fibroblastos/metabolismo , Regulación de la Expresión Génica/genética , ARN Mensajero/metabolismo , Animales , Embrión de Pollo , Citocinas/metabolismo , Femenino , Lipopolisacáridos , Mitosis/efectos de los fármacos , Conejos , Fase de Descanso del Ciclo Celular , Factor de Crecimiento Transformador beta/farmacología

RESUMEN

We isolated a cDNA for p20K, a secreted protein preferentially synthesized in nonproliferating cells. p20K mRNA and protein levels declined rapidly following treatment with various mitogens. DNA sequence analysis of the p20K cDNA predicted a novel protein distantly related to alpha 2 mu-globulin and plasma retinol-binding protein.

Asunto(s)

Proteínas Sanguíneas/genética , Factor de Crecimiento Epidérmico/farmacología , Insulina/farmacología , Proteínas de los Retroviridae/farmacología , Secuencia de Aminoácidos , Animales , Proteínas Aviares , Secuencia de Bases , Proteínas Sanguíneas/metabolismo , Células Cultivadas , ADN/genética , Regulación de la Expresión Génica , Interfase , Lipocalinas , Datos de Secuencia Molecular , Proteína Oncogénica pp60(v-src) , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

The Spec1 and Spec2 proteins of the sea urchin Strongylocentrotus purpuratus are related to calmodulin, troponin C, and myosin light chains by sequence similarity in their four calcium binding domains. These domains, the EF-hands, are distinct helix-loop-helix structures of about 40 amino acids. The Spec1 and Spec2 genes are expressed specifically in aboral ectoderm cells of the developing embryo; however, the function of the Spec proteins in these cells is unknown. To find conserved regions of the proteins that might be important for structure and function, Spec homologues from Lytechinus pictus, a distantly related sea urchin, were sought. L. pictus embryos do not synthesize detectable amounts of the 14,000-17,000-Da Spec proteins as determined by two-dimensional gel electro-phoresis, but do synthesize three 34,000-Da proteins that cross-react with Spec1 antibodies and display a similar ontogenetic pattern of expression. cDNA clones were isolated by hybridization to a synthetic oligonucleotide corresponding to the EF-hand. One clone, LpS1, encodes an mRNA with developmental properties like those of the S. purpuratus Spec mRNAs. However, LpS1 contains an open reading frame for a protein of 34,000 Da rather than 17,000 Da, and antibodies raised against part of the LpS1 reading frame demonstrate that LpS1 encodes a 34,000-Da protein in L. pictus embryos. The sequence of LpS1 reveals the presence of eight EF-hand domains, which share structural homology with the Spec1 or Spec2 EF-hands; however, little else in the protein sequence is conserved. The results support the hypothesis that the LpS1 gene arose from a duplication of an ancestral Spec gene and that the overall structural features of the Spec family of proteins are more conserved than the amino acid sequences.

Asunto(s)

Troponina/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/análisis , Datos de Secuencia Molecular , Peso Molecular , ARN Mensajero/metabolismo , Erizos de Mar , Troponina/genética , Troponina C

RESUMEN

We have completed the molecular analysis of a ribosomal protein S6 kinase identified in unfertilized Xenopus eggs. Although some of our results support the notion that an antigenically related enzyme is present in animal cells, we are uncertain if such an enzyme accounts for all of the mitogen-stimulated phosphorylation of 40S subunits. Other experiments have led to the identification of mRNAs that are rapidly expressed in mitogen-stimulated cells. The relevance of such genes and their products is in doubt, however, until a function can be demonstrated for each of them. Preliminary experiments with the pCEF-4 gene product described here have failed to show that it acts as a mitogen for cells in culture, for example. Thus, although studies such as these may lead to the identification of novel genes, we may need to search elsewhere for a physiologically significant function.

Asunto(s)

Regulación de la Expresión Génica , Genes , Proteínas Quinasas/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Línea Celular , Células Cultivadas , Femenino , Mitógenos , Datos de Secuencia Molecular , Proteína Oncogénica pp60(v-src) , Óvulo/enzimología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas de los Retroviridae/genética , Proteínas Quinasas S6 Ribosómicas , Sistemas de Mensajero Secundario/efectos de los fármacos , Homología de Secuencia de Ácido Nucleico , Xenopus

RESUMEN

A molecular clone corresponding to a 1.2-kilobase mRNA enriched in Rous sarcoma virus-transformed chicken embryo fibroblasts (CEF) was identified by differential screening of a cDNA library. The induction of the cloned sequence (denoted pCEF-4) in CEF infected by the temperature-sensitive mutant NY72-4 Rous sarcoma virus is rapid and independent of protein synthesis. DNA sequencing of the 1.2-kilobase insert of CEF-4 revealed an open reading frame that predicts an 11-kDa protein. The predicted pCEF-4 gene product is homologous to human connective tissue-activating peptide III (CTAP-III) and platelet factor 4 (PF-4). Serum stimulation of quiescent normal CEF results in a rapid but transient expression of pCEF-4 mRNA. Hence, pCEF-4 mRNA is expressed at the G0-G1 transition and during the first G1 phase of normal CEF reentering the cell cycle. The expression of pCEF-4 mRNA in Rous sarcoma virus-transformed CEF appears to be the result of transcriptional activation and stabilization of the transcript.

Asunto(s)

Virus del Sarcoma Aviar/genética , Proteínas Sanguíneas/genética , Transformación Celular Neoplásica , Genes , Péptidos/genética , Proteínas de los Retroviridae/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Embrión de Pollo , Clonación Molecular , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de Ácido Nucleico

RESUMEN

Chicken heart mesenchymal cells do not proliferate in medium of physiological composition containing plasma (S. Balk, Proc. Natl. Acad. Sci. USA 77:6606-6610, 1980). To understand the molecular events involved in cell quiescence and in the initiation of cell division under physiological conditions, we examined the differences in the patterns of protein synthesis of quiescent, hormone-stimulated, and Rous sarcoma virus-transformed chicken heart mesenchymal cells. We describe the expression of a 20,000-kilodalton (kDa) polypeptide actively synthesized by quiescent cells but not by their transformed counterparts. Normal chicken heart mesenchymal cells stimulated with epidermal growth factor and insulin also repressed the synthesis of the 20,000-kDa polypeptide while actively growing but synthesized increasing amounts of the protein at high cell density (confluence). The synthesis of the 20,000-kDa protein is not restricted to chicken heart mesenchymal cells, since confluent, density-arrested chicken embryo fibroblasts also expressed high levels of the protein. Transformed chicken heart mesenchymal cells and embryo fibroblasts did not synthesize the protein even at high cell density. The 20,000-kDa polypeptide accumulated in the culture medium.

Asunto(s)

Virus del Sarcoma Aviar/genética , Transformación Celular Neoplásica , Miocardio/metabolismo , Péptidos/genética , Animales , División Celular , Células Cultivadas , Pollos , Técnica del Anticuerpo Fluorescente , Sueros Inmunes , Masculino , Miocardio/citología , Péptidos/análisis

RESUMEN

Changes in protein synthesis induced by heat shock of Strongylocentrotus purpuratus gastrulae were analyzed bt two-dimensional electrophoresis. Hyperthermia induces the synthesis of polypeptides having molecular masses of 90, 70, 50, 40, and 38 kDa. One of these, hsp90, appears as a pair of polypeptides which comigrates with proteins synthesized at normal temperature in eggs and embryos; these comigrating spots produce indistinguishable patterns upon electrophoretic analysis of partial V8 protease digests, indicating that hsp90 is synthesized throughout embryogenesis. The relative rate of incorporation of methionine into hsp90 is low in eggs and zygotes, but increases abruptly in morulae, constituting a rare and striking change in protein synthesis during early development. Cell-free translation analyses indicate that most of the mRNA encoding hsp90 resides in the pool of free ribonucleoprotein particles in eggs and early embryos, but shifts to polysomes by the 64-cell stage while remaining constant in mass. Thus the increase in synthesis of hsp90 appears to be via the selective activation of translation of a stored maternal mRNA. The shift of hsp90 mRNA to polysomes is accompanied by polyadenylation. Heat shock of eggs or zygotes did not result in translational activation of hsp90 mRNA. The sea urchin hsp90 doublet of spots comigrates with hsp90 induced by heat shock of chicken embryo fibroblasts, a conserved protein abundant in many cells of a variety of species.

Asunto(s)

Proteínas de Choque Térmico/genética , Erizos de Mar/embriología , Animales , Regulación de la Expresión Génica , Proteínas de Choque Térmico/biosíntesis , Punto Isoeléctrico , Peso Molecular , Biosíntesis de Proteínas , ARN Mensajero/genética , Erizos de Mar/genética