Clinical aspects and diagnostic relevance of neuroautonomic evaluation in patients with unexplained falls.

OBJECTIVE: To evaluate the diagnostic relevance of neuroautonomic evaluation in patients with unexplained falls compared to those with a syncope etiologically unexplained after initial evaluation. METHODS: It is an observational study, comparing 298 patients with unexplained fall with 989 patients with unexplained syncope. Each patient underwent supine and upright blood pressure measurement, tilt testing (TT) and carotid sinus massage (CSM). RESULTS: Patients with unexplained falls were older (75.3 ± 11.1 vs. 63.2 ± 19.2 years, p < 0.001), were more frequently hypertensive (66.1 vs. 47.2 %, p < 0.001) and more frequently prescribed antihypertensive drugs (62.4 vs. 48.7 %, p < 0.001) or benzodiazepines (15.7 vs. 10.6 %, p = 0.01), and in a greater proportion they experienced major traumatic injuries (77.5 vs. 29.6 %, p < 0.001) as a consequence of falls. The TT was less frequently positive in patients with unexplained falls (36 vs. 51.3 %, p < 0.001), whereas a Carotid Sinus Syndrome as suggested by CSM had a similar prevalence in the two groups (14.3 vs. 10.5 %, p = 0.074). Overall, either TT or CSM were positive in 61 % of patients with unexplained falls, and in 64 % of those with syncope (p = 0.346). After matching by age 298 patients with falls (75.3 ± 11.1 years) and 298 patients with unexplained syncope (75.4 ± 11.1 years), we found that the positivity prevalence of TT and CSM were similar in the two groups. CONCLUSIONS: The positivity prevalence of TT and CSM in patients with unexplained falls compared to patients with unexplained syncope is similar. Given its high diagnostic relevance, the neuroautonomic evaluation should be routinely performed in older patients with unexplained falls.

The management of syncope in older adults.

Syncope is a frequent symptom in older patients. The diagnostic and therapeutic management may be complex, particularly in older adults with syncope and comorbidities or cognitive impairment. Morbidity related to syncope is more common in older persons and ranges from loss of confidence, depressive illness and fear of falling, to fractures and consequent institutionalization. Moreover, advan-ced age is associated with short and long-term morbidity and mortality after syncope. A standardized approach may obtain a definite diagnosis in more than 90% of the older patients with syncope and may reduce diagnostic tools and hospitalizations. The initial evaluation, including anamnesis, medical examination, orthostatic hypotension test and electrocardiogram (ECG), may be more difficult in the elderly, specially for the limited value of medical history, particularly for the certain diagnosis of neuro-mediated syncope. For this reason neuroautonomic assessment is an essential step to confirm a suspect of neuromediated syncope. Orthostatic blood pressure measurement, head up tilt test, carotid sinus massage and insertable cardiac monitor are safe and useful investigations, particularly in older patients. The most common causes of syncope in the older adults are orthostatic hypotension, carotid sinus hypersensitivity, neuromediated syncope and cardiac arrhythmias. The diagnostic evaluation and the treatment of cardiac syncope are similar in older and young patients and for this reason will not be discussed. In older patients unexplained falls could be related to syncope, particularly in patients with retrograde amnesia. There are no consistent differences in the treatment of syncope between older and younger population, but a specific approach is necessary for orthostatic hypotension, drug therapy and pacemaker implantation.

MITOSTATIN, a putative tumor suppressor on chromosome 12q24.1, is downregulated in human bladder and breast cancer.

Allelic deletions on human chromosome 12q24 are frequently reported in a variety of malignant neoplasms, indicating the presence of a tumor suppressor gene(s) in this chromosomal region. However, no reasonable candidate has been identified so far. In this study, we report the cloning and functional characterization of a novel mitochondrial protein with tumor suppressor activity, henceforth designated MITOSTATIN. Human MITOSTATIN was found within a 3.2-kb transcript, which encoded a approximately 62 kDa, ubiquitously expressed protein with little homology to any known protein. We found homozygous deletions and mutations of MITOSTATIN gene in approximately 5 and approximately 11% of various cancer-derived cells and solid tumors, respectively. When transiently overexpressed, MITOSTATIN inhibited colony formation, tumor cell growth and was proapoptotic, all features shared by established tumor suppressor genes. We discovered a specific link between MITOSTATIN overexpression and downregulation of Hsp27. Conversely, MITOSTATIN knockdown cells showed an increase in cell growth and cell survival rates. Finally, MITOSTATIN expression was significantly reduced in primary bladder and breast tumors, and its reduction was associated with advanced tumor stages. Our findings support the hypothesis that MITOSTATIN has many hallmarks of a classical tumor suppressor in solid tumors and may play an important role in cancer development and progression.

The role of the insulin receptor substrate-1 in the differentiation of rat hippocampal neuronal cells.

H19-7/IGF-IR cells are rat hippocampal cells expressing a human IGF-I receptor, which differentiate to a neuronal phenotype when stimulated by IGF-I at 39 degrees C. H19-7/IGF-IR cells have low levels of expression of insulin receptor substrate-l (IRS-1), a major substrate of the IGF-IR. IGF-I induces serine-phosphorylation and down-regulation of the endogenous IRS-1 upon differentiation of H19-7/IGF-IR cells. The profound influence of IRS-1 on differentiation of H19-7/IGF-IR cells was confirmed by transfecting these cells with a plasmid expressing mouse IRS-1. Over-expression of wild type IRS-1 in H19-7/IGF-IR cells abolishes IGF-I-induced differentiation at 39 degrees C. A mutant of IRS-1 lacking the PTB domain loses the ability to inhibit the differentiation program. H19-7/IGF-IR/IRS-1 cells at 39 degrees C show a stronger and prolonged activation of Akt, when compared to H19-7/IGF-IR cells. The role of Akt in the inhibition of the differentiation program was confirmed by using the inhibitor of Class I PI3 kinases LY29400, which restores IGF-I-induced differentiation of H19-7/IGF-IR/IRS-1 cells. H19-7/IGF-IR/IRS-1 cells show a strong reduction in MAP kinases signaling, which is related to the superactivation of Akt. This was confirmed by expressing in H19-7/IGF-IR cells a constitutively active Akt, which inhibited MAP kinases activation in these cells. These experiments confirm the importance of MAPK in the mechanism of IGF-I-mediated differentiation of H19-7/IGF-IR cells

Anti-apoptotic signaling of the insulin-like growth factor-I receptor through mitochondrial translocation of c-Raf and Nedd4.

The type 1 insulin-like growth factor receptor (IGF-IR) sends a strong anti-apoptotic signal by at least three different pathways. By using mutants of the IGF-IR, we showed that one of the pathways depends on residues of the IGF-IR (serines 1280--1283) that interact with 14.3.3 proteins. The result is the activation of Raf-1 and the mitochondrial translocation of both Raf-1 and Nedd4, a target of caspases. A mutant IGF-IR in which the serines at positions 1280--1283 have been mutated to alanine does not protect from apoptosis and fails to translocate Nedd4 or Raf-1 to the mitochondria. This failure is accompanied by a loss of cytochrome c from the mitochondria. The 14.3.3/Raf-1/Nedd4 pathway is operative in the presence or absence of the insulin receptor substrate-1.

Role of phosphatidylinositol 3-kinase-Akt pathway in nucleophosmin/anaplastic lymphoma kinase-mediated lymphomagenesis.

The NPM/ALK fusion gene, formed by the t(2;5) translocation in a subset of anaplastic large cell lymphomas, encodes a Mr 75,000 hybrid protein that contains the NH2-terminal portion of the nucleolar phosphoprotein nucleophosmin (NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK). NPM/ALK encodes a constitutively activated tyrosine kinase that belongs to the family of tyrosine kinases activated by chromosomal translocations. Our studies showed that NPM/ALK, similar to other members of this family, activates phosphatidylinositol 3-kinase (PI3K) and its downstream effector, serine/threonine kinase (Akt). PI3K was found in complex with NPM/ALK. Both PI3K and Akt kinase were permanently activated in NPM/ALK-transfected BaF3 murine hematopoietic cells and in NPM/ALK-positive, but not in NPM/ALK-negative, patient-derived anaplastic large cell lymphoma cell lines. In addition, Akt was phosphorylated/activated in protein samples isolated from four patients diagnosed with ALK-positive T/null-cell lymphomas. The PI3K inhibitors wortmannin and LY294002 induced apoptosis in NPM/ALK+ cells but exerted only minor effects on the control BaF3 parental cells and peripheral blood mononuclear cells stimulated by growth factors. Furthermore, retroviral infection of NPM/ALK+ BaF3 cells with a dominant-negative PI3K mutant (delta p85) or a dominant-negative Akt mutant (K179M) inhibited proliferation and clonogenic properties of the infected cells. Finally, the Akt mutant (K179M) suppressed the tumorigenicity of NPM/ALK-transfected BaF3 cells injected into syngeneic mice. In conclusion, our data indicate that NPM/ALK constitutively activates the PI3K-Akt pathway and that this pathway plays an important role in the NPM/ALK-mediated malignant transformation.

Regulation of Id2 gene expression by the insulin-like growth factor I receptor requires signaling by phosphatidylinositol 3-kinase.

The Id proteins play an important role in proliferation, differentiation, and tumor development. We report here that Id gene expression can be regulated by the insulin-like growth factor I receptor (IGF-IR), a receptor that also participates in the regulation of cellular proliferation and differentiation. Specifically, we found that the IGF-IR activated by its ligand was a strong inducer of Id2 gene expression in 32D murine hemopoietic cells. This activation was not simply the result of cellular proliferation, as Id2 gene expression was higher in 32D cells stimulated by IGF-I than in cells exponentially growing in interleukin-3. The up-regulation of Id2 gene expression was largely dependent on the presence of insulin receptor substrate-1, a major substrate of the IGF-IR and a potent activator of the phosphatidylinositol 3-kinase (PI3K) pathway. The role of PI3K activity in the up-regulation of Id2 gene expression by the IGF-IR was confirmed by different methods and in different cell types. In 32D cells, the up-regulation of Id2 gene expression by the PI3K pathway correlated with interleukin-3 independence and inhibition of differentiation.

IGF-I receptor signaling in a prostatic cancer cell line with a PTEN mutation.

LNCaP prostatic cancer cells are characterized by having a PTEN mutation, low levels of type 1 insulin-like growth factor receptor (IGF-IR) and no IRS-1, one of the major substrates of the IGF-IR. The absence of IRS-1, an activator of PI3-kinase, is compensated in these cells by the mutation in PTEN, an inhibitor of PI3-kinase. However, IGF-IR signaling in the absence of IRS-1 can cause cell differentiation and growth arrest. We hypothesized that these three characteristics may not be unrelated, specifically that, together, they may favor the metastatic spread of prostatic cancer cells without decreasing their growth potential. In support of this hypothesis, we report here that: (1) IRS-1 expression increases cell adhesion and decreases cell motility; (2) over-expression of the IGF-IR, in the absence of IRS-1, causes growth arrest and (3) a combination of IGF-IR and IRS-1 restores the transformed phenotype of LNCaP cells. These findings suggest a mechanism by which prostatic cancer cells can achieve metastatic potential without interfering with their growth potential. Oncogene (2000).

Insulin receptor substrate-1, p70S6K, and cell size in transformation and differentiation of hemopoietic cells.

After an initial burst of cell proliferation, the type 1 insulin-like growth factor receptor (IGF-IR) induces granulocytic differentiation of 32D IGF-IR cells, an interleukin-3-dependent murine hemopoietic cell line devoid of insulin receptor substrate-1 (IRS-1). The combined expression of the IGF-IR and IRS-1 (32D IGF-IR/IRS-1 cells) inhibits IGF-I-mediated differentiation, and causes malignant transformation of 32D cells. Because of the role of IRS-1 in changing the fate of 32D IGF-IR cells from differentiation (and subsequent cell death) to malignant transformation, we have looked for differences in IGF-IR signaling between 32D IGF-IR and 32D IGF-IR/IRS-1 cells. In this report, we have focused on p70(S6K), which is activated by the IRS-1 pathway. We find that the ectopic expression of IRS-1 and the inhibition of differentiation correlated with a sustained activation of p70(S6K) and an increase in cell size. Phosphorylation in vivo of threonine 389 and, to a lesser extent, of threonine 421/serine 424 of p70(S6K) seemed to be a requirement for inhibition of differentiation. A role of IRS-1 and p70(S6K) in the alternative between transformation or differentiation of 32D IGF-IR cells was confirmed by findings that inhibition of p70(S6K) activation or IRS-1 signaling, by rapamycin or okadaic acid, induced differentiation of 32D IGF-IR/IRS-1 cells. We have also found that the expression of myeloperoxidase mRNA (a marker of differentiation, which sharply increases in 32D IGF-IR cells), does not increase in 32D IGF-IR/IRS-1 cells, suggesting that the expression of IRS-1 in 32D IGF-IR cells causes the extinction of the differentiation program initiated by the IGF-IR, while leaving intact its proliferation program.

Insulin-like growth factor I receptor signaling in differentiation of neuronal H19-7 cells.

The type I insulin-like growth factor receptor (IGF-IR) is known to send two seemingly contradictory signals inducing either cell proliferation or cell differentiation, depending on cell type and/or conditions. H19-7 cells are rat hippocampal neuronal cells immortalized by a temperature-sensitive SV40 large T antigen that grow at 34 degrees C in epidermal growth factor or serum but differentiate at 39 degrees C when induced by basic fibroblast growth factor. At 39 degrees C, expression of the human IGF-IR in H19-7 cells induces an insulin-like growth factor (IGF) I-dependent differentiation. We have investigated the domains of the IGF-IR required for differentiation of H19-7 cells. The tyrosine 950 residue and serines 1280-1283 in the COOH terminus of the receptor are required for IGF-I-induced differentiation at 39 degrees C, although they are dispensable for IGF-I-mediated growth at 34 degrees C. Both domains have to be mutated to inactivate the differentiating function. The inability of these mutant receptors to induce differentiation correlates with mitogen-activated protein kinase activation. In contrast, inhibitors of phosphatidylinositol 3'-kinase have no effect on IGF-I-mediated differentiation of H19-7 cells, although they do inhibit the mitogenic response.

Domains of the insulin-like growth factor I receptor required for the activation of extracellular signal-regulated kinases.

The type 1 insulin-like growth factor receptor (IGF-IR) activates the extracellular signal-regulated kinases (ERK1 and -2). The two major substrates of the IGF-IR, insulin receptor substrate-1 (IRS-1) and the Shc proteins, are known to contribute to this activation. We investigated the domains of the IGF-IR required for the activation of the ERK proteins. To facilitate this study, we used a cell line (32D cells) that lacks IRS-1. In the absence of IRS-1, ERK activation is inhibited if the IGF-IR is mutated at two domains: tyrosine Y950 and a serine quartet at 1280-1283. Expression of IRS-1 in 32D cells expressing the double mutant IGF-IR restores ERK activation. The importance of the C-terminus of the IGF-IR in ERK activation (in the absence of IRS-1) is confirmed by the failure of the insulin receptor to give a sustained activation of ERK. In this model system, there is a good, but not exact, correlation between ERK activation and cell survival after withdrawal of growth factors.

Grb10 proteins in insulin-like growth factor and insulin receptor signaling (review).

The adapter protein Grb10 belongs to a superfamily of related proteins that include Grb7, Grb14 and the Caenorhabtidis elegans Mig10. The available data on Grb10 proteins suggest a role of these adapters in signaling downstream of several receptor tyrosine kinases. In the present review the focus is on the interaction between Grb10 proteins and the insulin-like growth factor receptor/insulin receptor, and the role of Grb10 in IGF-I/insulin-induced mitogenesis is discussed, considering that the data available are also partially discordant.

mGrb10 interacts with Nedd4.

We have utilized the yeast two-hybrid system to identify proteins interacting with mouse Grb10, an adapter protein known to interact with both the insulin and the insulin-like growth factor-I receptors. We have isolated a mouse cDNA clone containing the C2 domain of mouse Nedd4, a ubiquitin protein ligase (E3) that also contains a hect (homologous to the E6-AP carboxyl-terminus) domain and three WW domains. The interaction with Grb10 in the two-hybrid system was confirmed using the full-length Nedd4, and it was abolished by deleting the last 148 amino acids of Grb10, a region that includes the SH2 domain and the newly identified BPS domain. The interaction between Grb10 and Nedd4 was also reproduced in vivo in mouse embryo fibroblasts, where endogenous Nedd4 co-immunoprecipitated constitutively with both the endogenous and an overexpressed Grb10. This interaction was Ca(2+)-independent. Grb10 interacting with Nedd4 was not ubiquitinated in vivo, raising the possibility that this interaction may be used to target other proteins, like tyrosine kinase receptors, for ubiquitination.

Synthetic peptide sequence from the C-terminus of the insulin-like growth factor-I receptor that induces apoptosis and inhibition of tumor growth.

Although the type 1 insulin-like growth factor receptor (IGF-IR) is a potent inhibitor of apoptosis, its C-terminus sequence sends contradictory signals, including a clearly proapoptotic signal. We have synthesized a peptide, peptide 2, having the sequence of the IGF-IR from residue 1282 to residue 1298 (C-terminus of the beta subunit). To favor its uptake into cells, we linked it to a stearic acid moiety at its NH-terminus. Peptide 2 is taken up by the cells, where it inhibits DNA synthesis and causes apoptosis, while a scrambled peptide (with stearic acid) and peptide 2 without stearic acid are completely ineffective. Peptide 2 is more effective when cells are in anchorage-independent conditions than when they grow in monolayer cultures. Accordingly, we find that peptide 2 can inhibit the growth of a human prostatic cell line in nude mice. The proapoptotic effect of peptide 2 is inhibited by the expression of Bcl-2 or by a dominant negative mutant of caspase 9. These and other data indicate that peptide 2 does not seem to be competing directly with the IGF-IR for common substrates, but that its proapoptotic effect is related to its ability to activate the caspase cascade.

Growth and differentiation signals by the insulin-like growth factor 1 receptor in hemopoietic cells are mediated through different pathways.

The type 1 insulin-like growth factor receptor (IGF-IR) plays an important role in the growth of cells both in vivo and in vitro. The IGF-IR is also capable of inducing differentiation in a number of cell types, raising the question of how the same receptor can send two seemingly contradictory signals, one for growth and one for differentiation. Using 32D cells, which are murine hemopoietic cells, we show that the activated IGF-IR can induce differentiation along the granulocytic pathway in a manner similar to the granulocyte colony-stimulating factor. We find that one of the major substrates of the IGF-IR, the insulin receptor substrate-1 inhibits IGF-I-mediated differentiation of 32D cells. In the absence of insulin receptor substrate-1, functional impairment of another major substrate of the IGF-IR, the Shc proteins, is associated with a decrease in the extent of differentiation. Although the end points of the respective pathways remain to be defined, these results show for the first time that IGF-I-mediated growth or differentiation of hemopoietic cells may depend on a balance between two of its substrates.

Anti-apoptotic signaling of the IGF-I receptor in fibroblasts following loss of matrix adhesion.

The type 1 insulin-like growth factor receptor (IGF-IR) is known to protect cells from a variety of apoptotic injuries. In several instances, the anti-apoptotic effect of the wild type IGF-IR is more evident under conditions of anchorage-independence than in cells in monolayer cultures. We have investigated IGF-IR signaling in cells in anoikis, a form of apoptosis that occurs when cells are denied attachment to the extra-cellular matrix. IGF-I protects mouse embryo fibroblasts (MEF) from anoikis caused by withdrawal of growth factors. Survival is dependent on the concentration of IGF-I and a sufficient number of functional IGF-I receptors. In this model, IGF-I protection correlates best with ras activation and cell-to-cell aggregation, while PI3-kinase, Akt and MAP kinases seem to play a lesser, alternative role.

Differentiation and malignant transformation: two roads diverged in a wood.

Growth factors and their receptors are known to send at times contradictory signals, such as proliferation or differentiation. Recent developments in our knowledge of growth factor receptors and their signaling pathways have clarified the basis for this puzzling behavior. Separate domains of a receptor and/or the availability of specific substrates determine the fate of a cell stimulated by the same growth factor. In some models, the difference between malignant transformation and differentiation (leading to cell death) may depend on the presence or absence of a single agonist or antagonist molecule. The type 1 insulin-like growth factor receptor will serve as the paradigm for this review. J. Cell. Biochem. Suppls. 32/33:68-75, 1999.

The role of mGrb10alpha in insulin-like growth factor I-mediated growth.

Several isoforms of Grb10 are known to interact with either the insulin receptor or the insulin-like growth factor I (IGF-I) receptor, or both. Inasmuch as the data in the literature on the function of Grb10 are not always concordant, we have investigated the role of one of these isoforms, mGrb10alpha, in cell proliferation. For this purpose, a plasmid expressing mGrb10alpha was stably transfected into p6 cells and other mouse embryo fibroblast cell lines. An overexpressed mGrb10alpha inhibits IGF-I-mediated growth, causes a delay in the S and G2 phases of the cell cycle, and partially reverses the transformed phenotype. In contrast, it has no effect on insulin stimulation of cell proliferation. These studies indicate that this isoform of Grb10 has an inhibitory effect on IGF-I signaling of cell proliferation.

Insulin-like growth factor I receptor signaling in transformation by src oncogenes.

R- cells, a line of mouse embryo fibroblasts with a targeted disruption of the insulin-like growth factor I (IGF-I) receptor genes, are refractory to transformation by several viral and cellular oncogenes. Using colony formation in soft agar as a measure of full transformation, we report here that R- cells can be transformed by v-src, although they still cannot be transformed by the activated c-src527 (mutation at tyrosine 527 to phenylalanine), which readily transforms mouse embryo cells with a wild-type number of IGF-I receptors (W cells). Although v-src is a more potent inducer of tyrosine phosphorylation than c-src527, the extent of phosphorylation of either insulin receptor substrate 1 or Shc, two of the major substrates of the IGF-I receptor, does not seem sufficiently different to explain the qualitative difference in soft agar growth. v-src, however, is considerably more efficient than c-src527 in its ability to tyrosyl phosphorylate, in R- cells, the focal adhesion kinase, Stat1, and p130cas. These results indicate that v-src, but not c-src527, can bypass the requirement for a functional IGF-I receptor in the full transformation of mouse embryo fibroblasts and suggest that qualitative and quantitative differences between the two oncogenes can be used to identify some of the signals relevant to the mechanism(s) of transformation.