Respiratory alkalosis: no effect on blood lactate decline or exercise performance

It was the purpose of this study to determine the effects of respiratory alkalosis before and after high intensity exercise on recovery blood lactate concentration. Five subjects were studied under three different acid-base conditions before and after 45 s of maximal effort exercise: 1) hyperventilating room air before exercise (Respiratory Alkalosis Before = RALB, 2) hyperventilating room air during recovery (Respiratory Alkalosis After = RALA), and 3) breathing room air normally throughout rest and recovery (Control = C). RALB increased blood pH during rest to 7.65 +/- 0.03 while RALA increased blood pH to 7.57 +/- 0.03 by 40 min of recovery. Neither alkalosis treatment had a significant effect on blood lactate concentration during recovery. The peak lactate values of 12.3 +/- 1.2 mmol.L-1 for C, 11.8 +/- 1.2 mmol.L-1 for RALB, and 10.2 +/- 0.9 mmol.L-1 for RALA were not significantly different, nor were the half-times (t 1/2) for the decline in blood lactate concentration; C = 18.2 min, RALB = 19.3 min, and RALA = 18.2 min. In C, RALB and RALA, the change in base excess from rest to postexercise was greater than the concomitant increase in blood lactate concentration, suggesting the presence of a significant amount of acid in the blood in addition to lactic acid. There was no significant difference in either the total number of cycle revolutions (C = 77 +/- 2, RALB = 77 +/- 1) or power output at 5 s intervals between RALB and C during the 45 s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature-sensitive adult liver cell line dependent on glucocorticoid for differentiation.

A clonal rat adult hepatocyte cell line (RALA255-10G) was shown to be temperature sensitive (ts) for growth and differentiation. Glucocorticoid was necessary to maintain the maximal levels of differentiated functions in these cells. The RALA255-10G cell line was established by transforming primary adult hepatocytes with simian virus 40 tsA255 virus that is temperature sensitive for maintenance of transformation. At the permissive temperature (33 degrees C), RALA255-10G cells showed characteristics of malignant transformation, synthesized low levels of albumin and transferrin, and contained low levels of functional receptors for glucagon. At the nonpermissive temperature (40 degrees C), these cells regain the normal differentiated phenotype, and the levels of these three hepatic functions were increased. Induction of albumin and transferrin production by RALA255-10G cells at 40 degrees C was shown to be the result of the increase in the biosynthesis of these proteins. Furthermore, the albumin and transferrin produced by these cells were immunologically and electrophoretically indistinguishable from authentic rat albumin and transferrin. Glucocorticoid, which reduced the growth rate and saturation density of RALA255-10G cells at 33 degrees C, was absolutely required by these cells to synthesize albumin at both temperatures. This hormone also enhanced transferrin production and glucagon response. Our data indicate that glucocorticoid hormone is one of the factors that maintain adult hepatocytes in a differentiated state.     

Oncogenic Ras in tumour progression and metastasis

The ras genes give rise to a family of related GTP-binding proteins that exhibit potent transforming potential. Mutational activation of Ras proteins promotes oncogenesis by disturbing a multitude of cellular processes, such as gene expression, cell cycle progression and cell proliferation, as well as cell survival, and cell migration. Ras signalling pathways are well known for their involvement in tumour initiation, but less is known about their contribution to invasion and metastasis. This review summarises the role and mechanisms of Ras signalling, especially the role of the Ras effector cascade Raf/MEK/ERK, as well as the phosphatidylinositol 3-kinase/Akt pathway in Ras-mediated transformation and tumour progression. In addition, it discusses the impact of Rho GTPases on Ras-mediated transformation and metastasis.     

RhoE interferes with Rb inactivation and regulates the proliferation and survival of the U87 human glioblastoma cell line

Rho GTPases are important regulators of actin cytoskeleton, but they are also involved in cell proliferation, transformation and oncogenesis. One of this proteins, RhoE, inhibits cell proliferation, however the mechanism that regulates this effect remains poorly understood. Therefore, we undertook the present study to determine the role of RhoE in the regulation of cell proliferation. For this purpose we generated an adenovirus system to overexpress RhoE in U87 glioblastoma cells. Our results show that RhoE disrupts actin cytoskeleton organization and inhibits U87 glioblastoma cell proliferation. Importantly, RhoE expressing cells show a reduction in Rb phosphorylation and in cyclin D1 expression. Furthermore, RhoE inhibits ERK activation following serum stimulation of quiescent cells. Based in these findings, we propose that RhoE inhibits ERK activation, thereby decreasing cyclin D1 expression and leading to a reduction in Rb inactivation, and that this mechanism is involved in the RhoE-induced cell growth inhibition. Moreover, we also demonstrate that RhoE induces apoptosis in U87 cells and also in colon carcinoma and melanoma cells. These results indicate that RhoE plays an important role in the regulation of cell proliferation and survival, and suggest that this protein may be considered as an oncosupressor since it is capable to induce apoptosis in several tumor cell lines.     

Ion channels in cancer: future perspectives and clinical potential

Ion transport across the cell membrane mediated by channels and carriers participate in the regulation of tumour cell survival, death and motility. Moreover, the altered regulation of channels and carriers is part of neoplastic transformation. Experimental modification of channel and transporter activity impacts tumour cell survival, proliferation, malignant progression, invasive behaviour or therapy resistance of tumour cells. A wide variety of distinct Ca²⁺ permeable channels, K⁺ channels, Na⁺ channels and anion channels have been implicated in tumour growth and metastasis. Further experimental information is, however, needed to define the specific role of individual channel isoforms critically important for malignancy. Compelling experimental evidence supports the assumption that the pharmacological inhibition of ion channels or their regulators may be attractive targets to counteract tumour growth, prevent metastasis and overcome therapy resistance of tumour cells. This short review discusses the role of Ca²⁺ permeable channels, K⁺ channels, Na⁺ channels and anion channels in tumour growth and metastasis and the therapeutic potential of respective inhibitors.     

Ral GTPases regulate exocyst assembly through dual subunit interactions

Ral GTPases have been implicated in the regulation of a variety of dynamic cellular processes including proliferation, oncogenic transformation, actin-cytoskeletal dynamics, endocytosis, and exocytosis. Recently the Sec6/8 complex, or exocyst, a multisubunit complex facilitating post-Golgi targeting of distinct subclasses of secretory vesicles, has been identified as a bona fide Ral effector complex. Ral GTPases regulate exocyst-dependent vesicle trafficking and are required for exocyst complex assembly. Sec5, a membrane-associated exocyst subunit, has been identified as a direct target of activated Ral; however, the mechanism by which Ral can modulate exocyst assembly is unknown. Here we report that an additional component of the exocyst, Exo84, is a direct target of activated Ral. We provide evidence that mammalian exocyst components are present as distinct subcomplexes on vesicles and the plasma membrane and that Ral GTPases regulate the assembly interface of a full octameric exocyst complex through interaction with Sec5 and Exo84.     

Ras-induced transformation and signaling pathway.

Ras is a signal-transducing, guanine nucleotide-binding protein for various membrane receptors including tyrosine kinase receptors. Ras participates in the regulation of cell proliferation, differentiation, and morphology. Activated ras oncogenes have been identified in various forms of human cancer including epithelial carcinomas of the lung, colon, and pancreas. The cells of these cancers, as well as those that have been experimentally transformed by the activated ras gene, exhibit abnormal growth, morphological changes and alterations of cell adhesions. Although the main effector protein has been thought to be Raf serine/threonine kinase, research has revealed that the Ras-induced signaling pathway is mediated by multiple effector proteins and has the crosstalk with various factors containing other small GTPases. In this review, we summarize the involvement of each effector protein for Ras and the crosstalk with other small GTPases in Ras-induced transformation.     

Platelet-derived growth factor-C (PDGF-C) activation by serine proteases: implications for breast cancer progression

The PDGF (platelet-derived growth factor) family members are potent mitogens for cells of mesenchymal origin and serve as important regulators of cell migration, survival, apoptosis and transformation. Tumour-derived PDGF ligands are thought to function in both autocrine and paracrine manners, activating receptors on tumour and surrounding stromal cells. PDGF-C and -D are secreted as latent dimers, unlike PDGF-A and -B. Cleavage of the CUB domain from the PDGF-C and -D dimers is required for their biological activity. At present, little is known about the proteolytic processing of PDGF-C, the rate-limiting step in the regulation of PDGF-C activity. In the present study we show that the breast carcinoma cell line MCF7, engineered to overexpress PDGF-C, produces proteases capable of cleaving PDGF-C to its active form. Increased PDGF-C expression enhances cell proliferation, anchorage-independent cell growth and tumour cell motility by autocrine signalling. In addition, MCF7-produced PDGF-C induces fibroblast cell migration in a paracrine manner. Interestingly, PDGF-C enhances tumour cell invasion in the presence of fibroblasts, suggesting a role for tumour-derived PDGF-C in tumour-stromal interactions. In the present study, we identify tPA (tissue plasminogen activator) and matriptase as major proteases for processing of PDGF-C in MCF7 cells. In in vitro studies, we also show that uPA (urokinase-type plasminogen activator) is able to process PDGF-C. Furthermore, by site-directed mutagenesis, we identify the cleavage site for these proteases in PDGF-C. Lastly, we provide evidence suggesting a two-step proteolytic processing of PDGF-C involving creation of a hemidimer, followed by GFD-D (growth factor domain dimer) generation.     

Expression of CD40, CD44, bcl-2 antigens and rate of cell proliferation on fine needle aspirates from metastatic melanoma

The clinical behaviour of melanoma is often unpredictable using clinical and histological criteria. Tumour cell markers related to cell cycle regulation, apoptosis, cell-cell interactions and cell proliferation might improve the possibility of predicting the clinical course of melanoma. The aim of the present study was to refine prognostic criteria by an immunocytochemical investigation of CD44, CD40, bcl-2 antigens and cell proliferation in tumour cells aspirated from metastases of malignant melanoma. CD40 is a cell surface receptor shown to be expressed by lymphomas as well as carcinomas, and is thought to play a central role in the process of tumour progression. CD44 is a transmembrane glycoprotein, which is involved in growth signal transmission of importance in the binding of tumour cells to endothelium, cell migration and enhancement of cell motility, which makes it of interest to study in relation to the metastasizing capacity of tumours. The bcl-2 protein is active in the process of programmed cell death (apoptosis) as an antiapoptotic agent and its expression may reflect tumour progression. Mean/median percentages of tumour cell positivity were 8.5/3.0 for CD40, 76.1/86.3 for CD44 and 7.4/3.3 for bcl-2. A significant correlation was observed between expression of apoptosis-associated bcl-2 antigen and overall survival (r = 0.33). The CD44 positive cell fraction was higher in patients with short overall survival than those with long survival but this difference was not statistically significant. The expression of CD40 did not correlate with overall survival. The mean/median proliferation fraction assessed by MIB-1 monoclonal antibody was 25.8/23.9 and showed a significant correlation with survival after diagnosis of melanoma metastasis (r = 0.32). Lack of bcl-2 expression and a high proportion of tumour cells expressing Ki-67 antigen are predictors of poor prognosis that are independent of the traditionally accepted Breslow's thickness of the primary melanomas.     

Regulatory domain selectivity in the cell-type specific PKN-dependence of cell migration

The mammalian protein kinase N (PKN) family of Serine/Threonine kinases comprises three isoforms, which are targets for Rho family GTPases. Small GTPases are major regulators of the cellular cytoskeleton, generating interest in the role(s) of specific PKN isoforms in processes such as cell migration and invasion. It has been reported that PKN3 is required for prostate tumour cell invasion but not PKN1 or 2. Here we employ a cell model, the 5637 bladder tumour cell line where PKN2 is relatively highly expressed, to assess the potential redundancy of these isoforms in migratory responses. It is established that PKN2 has a critical role in the migration and invasion of these cells. Furthermore, using a PKN wild-type and chimera rescue strategy, it is shown that PKN isoforms are not simply redundant in supporting migration, but appear to be linked through isoform specific regulatory domain properties to selective upstream signals. It is concluded that intervention in PKNs may need to be directed at multiple isoforms to be effective in different cell types.     

Advances in Rho-dependent actin regulation and oncogenic transformation.

Cellular movement is central to invasion. The Rho family of small GTPases co-ordinate the cytoskeletal and adhesion modelling within cells that is crucial for normal migratory responses. Consequently, Rho proteins, and their regulators and effectors, are targets for subversion during oncogenic transformation and tumour development. Recent findings have thrown light on how actin regulators may be linked to oncogenesis and the development of cancer.     

Disruption of the Shc/Grb2 complex during abelson virus transformation affects proliferation, but not apoptosis

The v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) induces pre-B-cell transformation. Signals emanating from the SH2 domain of the protein are required for transformation, and several proteins bind this region of v-Abl. One such protein is the adaptor molecule Shc, a protein that complexes with Grb2/Sos and facilitates Ras activation, an event associated with Ab-MLV transformation. To test the role this interaction plays in growth and survival of infected pre-B cells, dominant-negative (DN) Shc proteins were coexpressed with v-Abl and transformation was examined. Expression of DN Shc reduced Ab-MLV pre-B-cell transformation and decreased the ability of v-Abl to stimulate Ras activation and Erk phosphorylation in a Raf-dependent but Rac-independent fashion. Further analysis revealed that Shc is required for v-Abl-mediated Raf tyrosine 340 and 341 phosphorylation, an event associated with Erk phosphorylation. In contrast to effects on proliferation, survival of the cells and activation of Akt were not affected by expression of DN Shc. Together, these data reveal that v-Abl-Shc interactions are a critical part of the growth stimulatory signals delivered during transformation but that they do not affect antiapoptotic pathways. Furthermore, these data highlight a novel role for Shc in signaling from v-Abl to Raf.     

RalB mobilizes the exocyst to drive cell migration

The Ras family GTPases RalA and RalB have been defined as central components of the regulatory machinery supporting tumor initiation and progression. Although it is known that Ral proteins mediate oncogenic Ras signaling and physically and functionally interact with vesicle trafficking machinery, their mechanistic contribution to oncogenic transformation is unknown. Here, we have directly evaluated the relative contribution of Ral proteins and Ral effector pathways to cell motility and directional migration. Through loss-of-function analysis, we find that RalA is not limiting for cell migration in normal mammalian epithelial cells. In contrast, RalB and the Sec6/8 complex or exocyst, an immediate downstream Ral effector complex, are required for vectorial cell motility. RalB expression is required for promoting both exocyst assembly and localization to the leading edge of moving cells. We propose that RalB regulation of exocyst function is required for the coordinated delivery of secretory vesicles to the sites of dynamic plasma membrane expansion that specify directional movement.