Biomedical subjects

G L Colucci-D'Amato

Publications and source records attributed to G L Colucci-D'Amato.

11 recordsLinked to original sources

PubMed2000

Abrogation of nerve growth factor-induced terminal differentiation by ret oncogene involves perturbation of nuclear translocation of ERK.

Oncogenic variants of the receptor tyrosine kinase, Ret, cause formation of tumors of neuroendocrine derivation in the multiple endocrine neoplasia type 2 and, thus, likely interfere with antiproliferative and/or differentiative extracellular signals. Here we took advantage of two rat pheochromocytoma-derived cell lines (PC12/MEN2A and PC12/MEN2B) to investigate whether Ret-induced nerve growth factor (NGF) unresponsiveness might involve impairment of ERK signaling. In fact, these cells, stably transfected with distinct forms of the active ret oncogene, fail to block proliferation, even upon NGF stimulation. In these cells we show the presence of both chronic ERKs activity and high expression levels of MKP-3, an ERK-specific phosphatase. Despite the presence of MKP-3, ERK activity can be further stimulated by NGF, but it fails to translocate into the nucleus and consequently to induce immediate-early gene transcription. Because of the presence of MKP-3, our results suggest the existence of a negative regulatory feedback acting on ERKs as a mechanism responsible for the abrogation of NGF-induced terminal differentiation. Indeed, MKP-3 seems to be implicated in the persistence of ERKs in cell cytoplasm. This interpretation is further supported by the observation that in ret-transfected cells, forced expression of an active form of MEK-1 may overcome this block; it restores transcription from the c-fos promoter, induces translocation of ERKs into the nucleus, and inhibits cell proliferation.

Animals↗

PubMed2000

Signaling through Ras is essential for ret oncogene-induced cell differentiation in PC12 cells.

Specific germline mutations of the receptor tyrosine kinase, Ret, predispose to multiple endocrine neoplasia types 2A and 2B and familial medullary thyroid carcinoma. The mechanisms by which different Ret isoforms (Ret-2A and Ret-2B) cause distinct neoplastic diseases remain largely unknown. On the other hand, forced expression of these mutated versions of Ret induces the rat pheochromocytoma cell line, PC12, to differentiate. Here we used an inducible vector encoding a dominant-negative Ras (Ras p21(N17)) to investigate the contributions of the Ras pathway to the phenotype induced in PC12 cells by the expression of either Ret-2A or Ret-2B mutants. We show that the Ret-induced molecular and morphological changes are both mediated by Ras-dependent pathways. However, even though inhibition of Ras activity was sufficient to revert Ret-induced differentiation, the kinetics of morphological reversion of the Ret-2B- was more rapid than the Ret-2A-transfected cells. Further, we show that in Ret-transfected cells the suc1-associated neurotrophic factor-induced tyrosine phosphorylation target, SNT, is chronically phosphorylated in tyrosine residues, and associates with the Sos substrate. These results indicate the activation of the Ras cascade as an essential pathway triggered by the chronic active Ret mutants in PC12 cells. Moreover, our data indicate SNT as a substrate for both Ret mutants, which might mediate the activation of this cascade.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Estradiol-induced mitogen-activated protein kinase (extracellular signal-regulated kinase 1 and 2) activity in the frog (Rana esculenta) testis.

Several lines of evidence support a key role of estradiol-17beta (E(2)) in male fertility. We have used a non-mammalian vertebrate model, the frog Rana esculenta, to investigate the regulation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) activity in the testis during the annual sexual cycle and to study whether E(2 )exerts a role in spermatogenesis through the regulation of ERK1/2 activity. ERK1/2 proteins are present in the cytoplasm and nucleus of the primary and secondary spermatogonia (SPG), and in the nucleus of primary spermatocytes. The annual E(2) profile shows a progressive increase during active spermatogenesis with a peak in the month of June. In parallel, ERK1/2 are highly phosphorylated during the period of active spermatogenesis (from April to July) compared with the regressive period (September/October) and winter stasis (from November to March). E(2) treatment induces the proliferation of primary SPG, possibly via the activation of ERK1/2, and this effect is counteracted by the anti-estrogen ICI 182-780.

Animals↗

PubMed1999

Neuronal and glial properties coexist in a novel mouse CNS immortalized cell line.

A mes-c-myc A1 (A1) cell line was generated by retroviral infection of cultured embryonic mesencephalic cells and selected by neomycin resistance. A1 cells cease to divide and undergo morphological differentiation after serum withdrawal or addition of c-AMP. Proliferating or morphologically differentiated A1 cells are all positive for vimentin and nestin, a marker of neural precursor, and show neuronal markers such as microtubule-associated protein 1, neuron-specific enolase and peripherin, and the glial marker glial fibrillary acidic protein. Neuronal and glial markers coexist in single cells. Furthermore, A1 cells show presence of glutamic acid decarboxylase 67 mRNA and its embryonic form EP10 and accumulate the neurotransmitter GABA. Electrophysiological studies demonstrate that morphologically differentiated A1 cells display voltage-gated sodium and potassium channels in response to depolarizing stimuli. A1 cells thus represent a novel, bipotent neural cell line useful for studying CNS differentiation and plasticity, as well as the molecular mechanisms underlying development of GABAergic neurotransmission.

Animals↗

PubMed1996

Ligand stimulation of a Ret chimeric receptor carrying the activating mutation responsible for the multiple endocrine neoplasia type 2B.

Inherited activating mutations of Ret, a receptor tyrosine kinase, predispose to multiple endocrine neoplasia (MEN) types 2A and 2B and familial medullary thyroid carcinoma. To investigate the effects induced by acute stimulation of Ret, we transfected both PC12 and NIH 3T3 cells with a molecular construct in which the ligand-binding domain of the epidermal growth factor receptor was fused to the catalytic domain of Ret. Acute stimulation of the chimeric receptor induced PC12 cells to express a neuronal-like phenotype. Moreover, we introduced the dominant mutation, responsible for the multiple endocrine neoplasia type 2B, in the catalytic domain of the Ret chimera. Expression of the mutant chimera, in the absence of ligand stimulation, induces the PC12 cells to acquire a flat morphology with short neuritic processes and transforms the NIH 3T3 cells. Stimulation of the mutant chimera with epidermal growth factor causes a drastic overgrowth of long neuritic processes, with the induction of the suc1-associated protein tyrosine phosphorylation in PC12 cells and higher transforming efficiency in NIH 3T3 cells. These data indicate that the gain-of-function MEN2B mutation does not abrogate ligand responsiveness of Ret and suggest that the presence of Ret ligand could play a role in the pathogenesis of the MEN2B syndrome.

3T3 Cells↗

PubMed1996

A potential pathogenetic mechanism for multiple endocrine neoplasia type 2 syndromes involves ret-induced impairment of terminal differentiation of neuroepithelial cells.

Germ-line missense mutations of the receptor-like tyrosine kinase ret are the causative genetic event of the multiple endocrine neoplasia (MEN) type 2A and type 2B syndromes and of the familial medullary thyroid carcinoma. We have used the rat pheochromocytoma cell line, PC12, as a model system to investigate the mechanism or mechanisms by which expression of activated ret alleles contributes to the neoplastic phenotype in neuroendocrine cells. Here we show that stable expression of ret mutants (MEN2A and MEN2B alleles) in PC12 cells causes a dramatic conversion from a round to a flat morphology, accompanied by the induction of genes belonging to the early as well as the delayed response to nerve growth factor. However, in the transfected PC12 cells, the continuous expression of neuronal specific genes is not associated with the suppression of cell proliferation. Furthermore, expression of ret mutants renders PC12 cells unresponsive to nerve growth factor-induced inhibition of proliferation. These results suggest that induction of an aberrant pattern of differentiation, accompanied by unresponsiveness to growth-inhibitory physiological signals, may be part of the mechanism of action of activated ret alleles in the pathogenesis of neuroendocrine tumors associated with MEN2 syndromes.

Adrenal Gland Neoplasms↗

PubMed1996

Presence of physiologically stimulated RET in adult rat brain: induction of RET expression during nerve regeneration.

The product of the RET proto-oncogene is a protein belonging to the receptor-like tyrosine kinase superfamily. RET is expressed in several neural crest-derived cell lineages and has been implicated in the correct development of the peripheral nervous system. To gain further insight into RET function, we investigated the presence of active RET in adult rat tissues. We show, by immunoblotting, that the products of the RET proto-oncogene (p155ret) are present in specific regions of adult rat brain, including the cerebellum, striatum, brainstem, hypothalamus, hippocampus, and olfactory bulb. Moreover, in the cerebellum, p155ret is phosphorylated in tyrosine residues, thus indicating that this brain structure contains p155ret in an activated state. Finally, the presence of RET in motoneurons prompted us to analyze the effects of hypoglossal nerve section on its expression. We observed a dramatic increase in p155ret in the motoneuron nuclei, thus suggesting that RET tyrosine kinase plays a role in the neuronal response to axotomy and/or during nerve regeneration.

Animals↗

PubMed1995

Dbl expression driven by the neuron specific enolase promoter induces tumor formation in transgenic mice with a p53(+/-) genetic background.

The dbl oncogene, generated by the truncation of the amino-terminal portion of the proto-oncogene sequence, encodes a guanine-nucleotide-releasing factor. The transforming activity of this oncogene has never been demonstrated in vivo or in vitro except in the NIH 3T3 mouse fibroblast cell line. The expression of the proto-dbl transcript is confined to tissues and tumors of neuroectodermal derivation. Therefore, to study the transforming activity of the dbl oncogene in vivo, we have generated transgenic mice that express this oncogene in neuroepithelial tissues. Mice carrying the dbl oncogene did not develop a tumor. Successively, to establish whether dbl interacts with the tumor suppressor gene p53 in tumorigenesis, we have used a p53 deficient mouse strain. The results reported here indicate that dbl is capable of causing tumor formation in vivo when its expression is driven in an appropriate cellular and genetic environment.

Animals↗

PubMed1991

Preferential expression of the dbl protooncogene in some tumors of neuroectodermal origin.

We have investigated the expression of the dbl protooncogene in a wide variety of human tumors of different embryological derivation. We found that proto-dbl mRNA could be detected preferentially in a few neoplastic histiotypes of neuroectodermal origin. The common, normal 5-kilobase size of the proto-dbl transcript detected indicated that the proto-dbl in these tumors was not rearranged. These data, in agreement with our previous reports, suggest that the dbl protooncogene is expressed in a highly tissue-specific manner and indicate that this gene may be involved in the growth and differentiation of some cells of neuroectodermal origin.

Animals↗

PubMed1990

Differential growth factor expression in transformed mouse NIH-3T3 cells.

The expression of growth factor-specific mRNA transcripts and the presence of biologically active growth factors in the conditioned medium and in the cell extracts from mouse NIH-3T3 cells transformed by different oncogenes (Ki-ras, mos, src, fms, fes, met, and trk), by a DNA tumor virus (SV40), or by a chemical carcinogen (N-nitrosomethylurea) were studied. In contrast to NIH-3T3 cells or simian virus 40 (SV40)-transformed 3T3 cells, all the other transformed NIH-3T3 cell lines expressed a 4.5 kb transforming growth factor-alpha (TGF alpha)-specific mRNA transcript and secreted immunoreactive and biologically active TGF alpha ranging from 100 to 225 ng/10(8) cells/48 h. In addition, in the transformed cell lines that were secreting elevated levels of biologically active TGF alpha, there was a 75-95% reduction in the total number of epidermal growth factor receptors on these cells. A 2.6 kb TGF beta mRNA transcript and TGF beta protein in the conditioned medium (30-140 ng/10(8) cells/48 h) was also detected in those lines expressing TGF alpha. Basic fibroblast growth factor-like activity (11-50 ng/10(8) cells) was detected in the cell lysates from NIH-3T3 cells transformed with N-nitrosomethylurea or with trk, where expression of specific 6.9 and 3.9 kb mRNA transcripts for basic fibroblast growth factor could also be found. B chain (c-sis) expression of platelet-derived growth factor was present only in trk-transformed NIH-3T3 cells in which specific c-sis 6.5 and 4.6 kb transcripts were identified. In contrast, platelet-derived growth factor A chain expression of 2.9 and 2.3 kb transcripts was found in ras-, met-, mos-, and fms-transformed NIH-3T3 cells. These results suggest that the expression of different sets of growth factors is controlled in part by structurally distinct groups of transforming genes.

Animals↗

PubMedDate not supplied

Preferential expression of the dbl proto-oncogene in some neuroectodermal tumors.

The dbl oncogene belongs to a unique class of human transforming genes. The dbl proto-oncogene is activated by substitution of the 5' portion of the gene with an unrelated human sequence. The proto-oncogene product is distributed between the soluble and membrane fractions of the cytoplasm and its function remains still unknown. In order to understand the biological role of dbl in human malignancies or during cell differentiation we have investigated the expression of the dbl oncogene in a wide number of human tumors of different embryological derivation. We found that dbl is preferentially expressed in a few neoplastic histiotypes of neuroectodermal origin. The transcript size of 5.3 Kb strongly suggests that the gene is not truncated in these tumors. These data, together with the information that the proto-oncogene has been found expressed in normal brain and adrenal medulla, indicate that dbl expression may be involved in cell differentiation of some tissues of neuroectodermic origin.

Gene Expression Regulation, Neoplastic↗