Biomedical subjects

R Comolli

Publications and source records attributed to R Comolli.

At least 37 records · Page 2Linked to original sources

PubMed1994

Membrane and nuclear protein kinase C activation in the early stages of diethylnitrosamine-induced rat hepatocarcinogenesis.

The activity and expression of Ca(2+)-dependent cPKC alpha and beta isoenzymes in the particulate, soluble (cytosolic) and nuclear fractions of rat liver and the expression of Ca(2+)-independent nPKC delta and aPKC zeta were examined during the early stages (30 and 60 min, 24 and 96 h and 7 and 60 days post-hepatectomy) of the Solt-Farber 'resistant hepatocyte' model of diethylnitrosamine (DENA)-induced hepatocarcinogenesis in Fischer 344 rats and related to the presence of gamma-glutamyl transpeptidase (GGT)-positive hyperplastic cell foci and persistent nodules in rat liver. Total PKC activity was unmodified by the carcinogenic treatment. In contrast, the PKC activity in the particulate, as well as nuclear fractions increased with time, reaching a maximum 60 days post-hepatectomy, with a decrease in the cytosolic activity. In carcinogen-treated animals maximal expression of cPKC alpha and beta isoenzymes was present 7 days post-hepatectomy, while no changes in nPKC delta and aPKC zeta immunoreactivity were detected. In the nucleus, no cPKC alpha isoform expression was observed, the cPKC beta expression being maximal at 60 days. Seven and 60 days post-hepatectomy GGT-positive hyperplastic cell foci and persistent nodules were present in rat liver respectively. Taken together, the results of this study suggest a role for nuclear cPKC beta and for cPKC alpha in promoting the selective growth of carcinogen-initiated hepatocytes in rat liver. No evidence for a role of Ca(2+)-independent nPKC delta and aPKC zeta isoenzymes in the early stages of DENA-induced liver carcinogenesis could be demonstrated.

Animals↗

PubMed1993

Biochemical and immunological characterization of calcium-dependent and -independent PKC isoenzymes in renal ischemia.

The PKC alpha, beta, delta, epsilon and zeta isoenzyme changes in the rat kidney cortex have been investigated after temporary occlusion of the renal artery, followed by reperfusion for up to 4 h. Increased PKC alpha, delta and epsilon immunoreactivity and PKC alpha activity occurred within 60 min of postischemic reflow, while PKC beta and zeta were unmodified. Increased PKC beta activity was present at later times of reflow (120 min), suggesting that the initial events of the regenerative repair response after an ischemic insult implicate the activation of specific PKC isoenzymes (alpha, delta, epsilon), playing a role in the control of cell proliferation.

Animals↗

PubMed1993

Analysis of calcium-dependent protein kinase C isoforms in the early stages of diethylnitrosamine-induced rat hepatocarcinogenesis.

The profiles of the calcium-dependent protein kinase C (PKC) isozymes alpha, beta, and gamma were examined in subcellular fractions from Fischer 344 rat liver during the early stages (48 h, 96 h, 7 d, and 60 d) of diethylnitrosamine (DEN)-induced carcinogenesis, using the Solt-Farber "resistant hepatocyte" model (DEN-2-acetylaminofluorene-partial hepatectomy; DEN-AAF-PH), and then related to the presence of focal or nodular gamma-glutamyl transpeptidase (GGT)-positive morphologic changes in the liver. After DEAE and hydroxyapatite column chromatography, two peaks, immunologically identified as PKC-alpha and -beta isoforms, were detected in the liver of normal (alpha/beta ratio = 4.0) and treated rats. In DEN-AAF-PH hepatocarcinogenesis an increase in PKC-alpha expression was found after PH (+43 +/- 19% at 48 h, alpha/beta ratio = 5.1; +125 +/- 25% at 96 h, alpha/beta ratio = 4.8), whereas the PKC-beta isoform appeared less significantly modified (+11 +/- 3% at 48 h and +89 +/- 17% at 96 h). Seven and 60 days after PH, a marked increase in the PKC-alpha (+96 +/- 20% and +150 +/- 48%, respectively) and PKC-beta isoforms (+158 +/- 41%, alpha/beta ratio = 3.1 and +130 +/- 26%, alpha/beta ratio = 4.4, respectively), occurred along with the appearance of GGT-positive altered hepatic foci and nodules in the liver sections. Sham hepatectomy caused PKC-alpha and -beta isoform activities similar to those of normal controls. In contrast, saline-AAF-PH-treated rats had downregulation of PKC-alpha after PH (alpha/beta ratio = 1.8 at 96 h), possibly due to the mitoinhibitory effect of the carcinogen AAF on normal uninitiated hepatocytes. Immunohistochemical analysis with monoclonal antibodies to PKC-alpha and -beta revealed diffuse positive cytoplasmic signals in GGT-positive foci and nodules in rat liver. Taken together, these preliminary results, using the Solt-Farber model of liver carcinogenesis, suggest a role for PKC in tumor promotion. They also suggest that the PKC-alpha isoform may play a specific role in clonal expansion of DEN-initiated hepatocytes after PH.

2-Acetylaminofluorene↗

PubMed1993

Ribosomal protein S6 kinase and protein kinase C activation by epidermal growth factor after temporary renal ischemia.

An initial event in cell response to growth factors is the change in the state of phosphorylation of a number of cellular proteins playing a role in cell proliferation. The effects of a single dose of exogenously administered epidermal growth factor (EGF) on renal serine/threonine protein kinases such as ribosomal protein S6 kinase(s) and protein kinase C (PKC) and on [3H]thymidine incorporation into tubule cell nuclei have been studied during the regenerative repair response after temporary renal ischemia in the rat, followed by reperfusion for up to 72 h. During the postischemic reflow, the PKC and S6 kinase activities increased at 24 and 72 h, respectively. EGF anticipated both increases: the PKC at 4 and the S6 kinase(s) at 24 h. Associated with this EGF-induced rise of S6 kinase activity, a significant increase in renal tubule cell proliferation was observed. These studies suggest the presence of a growth factor-activated serine/threonine phosphorylation cascade in the rat kidney participating in the regulation of cell growth during recovery from an ischemic insult.

Animals↗

PubMed1992

Changes in alanine transport in plasma membrane vesicles from rat liver during the early stages of diethylnitrosamine-induced hepatocarcinogenesis.

The transport of L-alanine, a natural substrate of system A, across liver plasma membrane vesicle preparations was modified during the early stages of rat DENA hepatocarcinogenesis. Kinetic studies indicated an increase of the Vmax, with normal Km values, at 30 h in rats undergoing a partial hepatectomy. Normal Vmax and drastically reduced Km values were present using membrane preparations from liver tissue showing enzyme-altered hyperplastic foci and/or preneoplastic nodules. The results suggest that alanine transport is differently affected by initiating and promoting stimuli during rat DENA hepatocarcinogenesis. The changes of the Vmax could be related to the promoting effect of partial hepatectomy on cell proliferation whereas the changes of the affinity constant (Km) could be the result of intrinsic modifications of the transporter in initiated cells.

2-Acetylaminofluorene↗

PubMed1992

Ribosomal protein S6 kinase is activated after folic acid injury and epidermal growth factor administration but not after unilateral nephrectomy in the rat kidney.

Following loss of functional renal mass induced by a single parenteral injection of folic acid, the increased proliferation of tubular epithelium to replace injured and necrotic cells was associated with S6 kinase activation and enhanced phosphorylation of this ribosomal protein in vitro, EGF administered 1.5 h after folic acid promoted the phosphorylation of S6 protein and accelerated the regenerative repair process. In contrast, the hypertrophic response of the kidney following uninephrectomy was not associated with S6 kinase activation. The results of this study suggest a close correlation between levels of S6 protein phosphorylation and the degree of cell proliferation in the rat kidney.

Animals↗

PubMed1990

Alanine transport in plasma membrane vesicles from Yoshida ascites hepatoma cells (AH 130) in the exponential and stationary phase of growth.

The Na(+)-dependent transport of L-alanine into plasma membrane vesicles from Yoshida ascites hepatoma (AH-130) cells in the exponential and stationary phase of growth has been studied. A transient accumulation of the amino acid occurred in the presence of an inwardly directed sodium gradient, in both conditions. However, the height and the shape of the overshoot curve differed noticeably in the two preparations. The accumulation ratio increased three-fold and the maximal uptake value occurred at an earlier time in plasma membrane vesicles from exponential growing rather than stationary phase cells. This might suggest that one of the two systems, A or ASC, serving hepatocytes, is fully expressed only in the exponential phase of growth or, alternatively, that the kinetic parameters of a possibly unique transport system are modified. Inhibition, countertransport as well as adaptive stimulation experiments and kinetic studies suggested the presence of a unique carrier-mediated transport of alanine in both phases of growth. The Vmax value was drastically reduced in the stationary phase of growth whereas the Km value was almost the same in both preparations. Therefore, the differences in time courses observed could be related to changes of the Vmax of a single transport agency rather than to the appearance/disappearance of an additional transport system (e.g. system A) in the conditions studied.

Alanine↗

PubMed1990

Measurement with microelectrodes of intracellular Na+, K+, H+ and Cl- activities and of membrane potential in normal rat liver slices and during the 4-dimethylaminoazobenzene-induced rat hepatocarcinogenesis.

Steady-state membrane potentials (Vm) and intracellular Na+ (aiNa), K+ (aiK), H+ (aiH) and Cl- (aiCl) activities were measured with double-barrelled ion-selective microelectrodes in liver slices from normal rats and during the 4-dimethylaminoazobenzene-induced (DAB) hepatocarcinogenesis. Rats fed with the experimental regimen without the carcinogen were used as control animals. In Krebs-Henseleit bicarbonate saline containing 5.5 mM glucose as bathing solution at 37 degrees C, Vm was found to be significantly lower in neoplastic hepatocytes, compared to normal liver cells. Vm decreased also in control rat liver cells. Increased Na+/K+ ratios and Na+ + K+ activities were found in cancerous hepatocytes whereas H+ and Cl- activities decreased. Therefore, the intracellular pH increased significantly in neoplastic cells, compared to normal and control cells. This could reflect activation of the Na+/H+ exchange system during the DAB-induced hepatocarcinogenesis, leading to a stimulation of cell metabolism with increased rate of protein and DNA synthesis and loss of growth control, under these conditions.

Animals↗

PubMed1989

Activation of a ribosomal S6 protein kinase in rapidly emerging diethylnitrosamine-induced gamma-glutamyltranspeptidase-positive hyperplastic liver lesions of the rat.

The gamma-glutamyl transpeptidase (GGT)-positive hyperplastic liver lesions which developed in the Fisher 344 rat 7 and 60 days following a single carcinogenic dose of diethylnitrosamine (DENA, 200 mg/kg body weight), short-term dietary exposure to 0.02% 2-acetylaminofluorene (AAF) to suppress the growth of normal hepatocytes, and partial hepatectomy to actuate rapid growth of DENA altered hepatocytes not suppressed by AFF, showed an increased activity of a kinase which specifically phosphorylates the ribosomal S6 protein in vitro. Sham-operated animals showed, on the contrary, no GGT-positive cells and low S6 kinase activity, under the same conditions. After partial hepatectomy, activation of S6 kinase and elevated levels of phosphorylated S6 protein in vitro were detected in the early phases of "normal" hepatocyte proliferation, during liver regeneration, in DENA-treated, GGT-negative preparations, when the "selection" agent AAF was omitted from the diet. The observed activation of S6 kinase in GGT-positive hepatocytes and/or liver nodules could represent an early manifestation of the enhanced proliferation of altered hepatocytes during tumor induction and/or promotion under these conditions.

Animals↗

PubMed1988

Activation of a ribosomal protein S6 kinase during the 4-dimethylaminoazobenzene-induced rat hepatocarcinogenesis.

During the induction of 4-dimethylaminoazobenzene (DAB) hepatocarcinogenesis there was a progressive increase in the activity of a kinase which specifically phosphorylated S6 ribosomal protein in vitro. Liver tumour tissue (hepatocellular carcinoma and cholangioma) showed maximal activation of this kinase and elevated levels of S6 phosphorylation. Control rats fed with the same diet containing no dye showed a gradual decline of this activity, with low or lacking phosphorylation at the end of the experimental regimen, suggesting close correlation between tumor cell growth and phosphorylation of this ribosomal protein, under these conditions.

Animals↗

PubMed1988

Histidine transport in plasma membrane vesicles from rat liver.

The transport of L-histidine, a selective substrate for system N, across liver plasma membrane vesicles has been studied. The amino acid is accumulated sevenfold within the intravesicular space in the presence of a sodium gradient. Lithium can replace sodium to some extent in the cotransport mechanism. The amino acid translocation is not influenced by the transmembrane electrical potential difference. Histidine kinetics involves a saturable component plus a linear one both in the presence and in the absence of sodium. Sodium affects mainly the affinity of the translocator and to a lesser extent its mobility. Histidine uptake is competitively inhibited by glutamine and it is affected by alanine in a noncompetitive manner.

Alanine↗

PubMed1988

Heat shock, protein synthesis and ribosomal protein S6 phosphorylation in vitro in Yoshida AH 130 ascites hepatoma cells.

An activated 40S ribosomal protein S6 kinase has been demonstrated previously in cytosolic extracts from proliferating as well as resting cells of a very undifferentiated rat ascites hepatoma cell line (Yoshida AH 130), grown in vivo (Cell Biol. Int. Rep., 1986, 10, 821-831). In the present report we present evidence of unmodified activity of this kinase and S6 phosphorylation in vitro in cells submitted to a physiological stress such as a sublethal temperature elevation (heat shock: 42 degrees C for 2 h). The heat treatment causes a progressive decline in the number of active ribosomes and of L-35S methionine incorporation into total protein, suggesting drastically decreased synthesis of cellular proteins under these conditions. Cells recovering from heat shock show the induced synthesis of a protein with an apparent Mr of 50 kDa. Spontaneous high expression of heat shock proteins (HSP 70, 89, 100), without heat shock, occurs in these tumor cells.

Animals↗

PubMed1986

Protein synthesis, ribosomal protein S6 phosphorylation in vitro and the effects of amiloride: SDS gel electrophoresis studies in the Yoshida ascites tumor (AH 130) grown in vivo.

Cell-free cytosolic extracts from the Yoshida (AH 130) rat ascites hepatoma cell line, grown in vivo, showed high ribosomal protein S6 kinase activity in vitro, as measured by transfer of 32P to exogenous 40S rat liver ribosomal subunits, in both exponential growing and stationary phase cells. A significant decrease of protein synthesis (3H-leucine incorporation into total cell protein) was found to occur in cells reaching the stationary phase of growth, suggesting that S6 phosphorylation was not tightly coupled to the rate of the intraperitoneal cell growth and of protein synthesis in these tumor cells. When the cell-free cytosolic extracts were prepared from cells exposed to amiloride, at concentrations that inhibit the Na+/H+ exchange, a decrease of S6 kinase activity was observed only in exponential growing cells, suggesting the possibility of coupling of the Na+/H+ exchange with phosphorylation of intracellular proteins in these tumor cells. Actually, stationary phase cells showed unchanged S6 kinase activity under the same conditions, possibly due to the extremely low Na+/H+ exchange activity, previously demonstrated (Cell Biol. Int. Rep., 1985, 9, 1017-1025). The present experiments support the hypothesis that the regulation of protein synthesis is not tightly coupled to phosphorylation-dephosphorylation cycles, at least of ribosomal protein S6, in cells characterized by a rather uncontrolled growth such as the Yoshida (AH 130) rat ascites hepatoma. In this connection, an elevated degree of protein phosphorylation, such as that of the ribosomal protein S6, could be a general phenomenon of neoplastic transformation.

Amiloride↗

PubMed1985

Amiloride inhibits protein synthesis and lowers the intracellular pH in exponential growing Yoshida rat ascites hepatoma (AH 130) cells: evidence for a role of the Na+/H+ exchanger.

We have previously demonstrated in a rat ascites hepatoma cell line (Yoshida AH 130) the presence of a glucose-activatable and amiloride sensitive Na+/H+ exchange (Cell Biol. Int. Rep., 1984, 8, 297-307). Amiloride is known to inhibit this exchange and to cause a cytoplasmic acidification, with inhibition of protein and DNA synthesis, in cells induced to grow. Amiloride appears also to penetrate the cells and to inhibit directly protein synthesis. In the present report we describe experiments in which the activity of amiloride (0.1, 0.4 and 3.0 mM) on protein synthesis and the internal pH of cells was compared in exponential growing and stationary phase Yoshida ascites cells. In phosphate buffered medium and Na+ out = 147 mM no inhibition of protein synthesis (3H-leu incorporation into total cell protein) and no internal acidification (14C-DMO distribution between intra- and extracellular volume) were produced by 0.1 and 0.4 mM amiloride in exponential growing cells. In stationary phase cells, on the contrary, 0.4 mM amiloride inhibited protein synthesis by 60% without decreasing the internal pH. When the Na+ out was lowered to 25 mM, to reduce competition with amiloride, and/or all Na+ out was substituted with choline, 0.1 and 0.4 mM amiloride markedly inhibited protein synthesis and decreased the internal pH in exponential growing cells. No apparent inhibition occurred in stationary phase cells under the same conditions, possibly due to a preexistent internal acidification, with severe decrease of protein synthesis. Fluorimetric studies of amiloride "binding" to ascites cells showed that a reduced number of amiloride receptor sites could exist in Yoshida hepatoma cells at the stationary phase of growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗

PubMed1984

Amiloride and glucose effects on the intracellular pH of Yoshida rat ascites hepatoma AH-130 cells grown in vivo.

The equilibrium distribution of 5,5-dimethyloxazolidine-2,4-dione (DMO) between intra- and extracellular volume was used to estimate the intracellular pH in Yoshida rat ascites hepatoma AH-130 cells under different growth conditions (log, midlog and stationary). The cells were suspended in a Krebs-Ringer 25 mM phosphate buffer and the effects of variation of external pH, of glucose and amiloride addition on intracellular pH were measured. Proliferating cells had higher intracellular pH than stationary phase cells and this difference was inhibited by amiloride. On addition of glucose the fall in external pH was similar in all conditions and corresponded to lactate production. However, the intracellular pH decreased only in proliferating cells. Stationary phase cells showed an amiloride-sensitive cytoplasmic alkalinization with glucose. Glucose addition also caused prompt recovery to a normal polysomal pattern in these cells that might suggest increased efficiency of the initiation step of protein synthesis under these conditions. The data thus suggest that the increased intracellular pH of proliferating and of glucose-treated stationary phase cells is linked to the rate of protein synthesis and is mediated by the amiloride-sensitive Na+/H+ exchange system. This could lead to increased intracellular Na+ concentration under these conditions and to initiation of growth.

Amiloride↗

PubMed1983

Synthesis of native 60S and 40S ribosomal subunits in Yoshida rat ascites hepatoma AH-130 cells: correlation with the rate of cell growth.

The 60Sn and 40Sn subunit ribosome synthesis declined significantly in Yoshida rat ascites hepatoma AH-130 cells from the log phase to the plateau phase of the in vivo growth. Two main classes of 40Sn particles with protein/RNA ratios of 1.82 (p2) and 1.20 (p3) and a minor "heavy" one with protein/RNA ratio of 0.96 (p1) could be distinguished reproducibly by their ultraviolet absorption after sucrose zone sedimentation. The p2 particles appeared the dominating class in log phase cells. In plateau phase cells a decrease of p2 and an increase of p3 particles was observed. Under these conditions the p1 particles and the peaks corresponding to 60Sn subunits and to 80S ribosomes were also increased. Newly synthetized 40Sn particles banded in the p3 region of the gradient and p2 particles originated from them. These particles entered into the ribosomal cycle and contained poly(A) RNA. Formation of radioactive 80S couples by subunits entering into the ribosomal cycle was markedly stimulated in log phase cells and almost completely blocked in cells at the plateau phase of growth.

Animals↗

PubMed1982

Systemic IFN-alpha treatment of multiple bladder papilloma grade I or II patients: pilot study.

Bladder papillomatosis offers a good target to evaluate IFN-alpha systemic treatment. We carried out a pilot study on eight multiple bladder papilloma patients under the same treatment scheme (1 x 10(6) IU/amp. every 48 h over six months), and they were followed-up for over two years after treatment. Recurrent patients underwent a similar second treatment. IFN-alpha therapy showed the following variations of effects: total disappearance, size decrease or persistence of papillomas, neither size increase nor appearance of new ones, remarkable valuable recurrence frequency rate decrease in all cases, and recurrences with smaller papillomas. This IFN-alpha treatment scheme would be fit to carry out broader controlled studies to show frequencies of the different kinds of responses. The inclusion of a minimum (dose-frequency-period) IFN-alpha treatment period after the first six months' therapy is proposed in order to achieve total disappearance of recurrences.

Aged↗

PubMed1982

eIF-2 initiation factor activity in Yoshida ascites hepatoma AH 130 cells and in 4-dimethylaminoazobenzene-induced liver tumor tissue during growth.

Eukaryotic initiation factor 2 (eIF-2) preparations from mid-log and plateau-phase Yoshida ascites hepatoma AH 130 cells, from the liver of Yoshida ascites tumor-bearing rats and from 4-dimethylaminoazobenzene (DAB)-induced liver tumor tissue were assayed for ternary complex formation with 3H-met-tRNAf and GTP on nitrocellulose filters. The eIF-2 factor was extracted from postnuclear homogenate supernatants by high-salt wash and purified by ion exchange chromatography on DEAE-cellulose and phosphocellulose. The results here reported demonstrate changes of 3H-met-tRNAf X eIF-2 X GTP ternary complex formation under the conditions studied. Higher rates of ternary complex formation are present in control rat liver and in DAB-induced liver tumor tissue. The liver of Yoshida ascites tumor-bearing rats and the Yoshida ascites hepatoma cells show reduced rates of ternary complex formation, that are mostly evident at the plateau-phase of the intraperitoneal ascites cell growth. The present observations may be attributable to changes in the growing conditions of plateau-phase ascites cells, with accumulation in Gl phase, affecting the ability of eIF-2 to enter in the sequential assembly of the eukaryotic protein synthesis initiation complex.

Animals↗