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Biomedical subjects

A Franchi

Publications and source records attributed to A Franchi.

At least 145 records · Page 8Linked to original sources
PubMed1984

Blockade of the Na+/H+ antiport abolishes growth factor-induced DNA synthesis in fibroblasts. Structure-activity relationships in the amiloride series.

We have previously characterized in Chinese hamster lung fibroblasts a growth factor activatable and amiloride-sensitive Na+/H+ antiport (Pouysségur, J., Chambard, J. C., Franchi, A., Paris, S., and Van Obberghen-Schilling, E. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3935-3939). In this report, we compared the affinity of 28 analogs of amiloride for inhibition of the Na+/H+ antiport and inhibition of growth factor-induced DNA synthesis. We showed that the guanidino moiety of amiloride must be protonated to elicit inhibition of the Na+/H+ exchange. Substitutions within this moiety by methyl, phenyl, or benzyl groups reduced the activity 20- to 1000-fold. On the contrary, substitution of the proton(s) of the 5-amino group of amiloride with alkyl or alkenyl groups increases potency up to 100-fold (5-N,N-diethylamiloride has a KI of 4 X 10(-8) M). In HCO-3-free medium and at lower [Na+]0 (25 or 50 mM) to reduce competition with amiloride, we found that growth factor-stimulated DNA synthesis of G0-arrested cells is inhibited by amiloride and its analogs with the same rank order as that for Na+/H+ antiporter inhibition. Over a range of 3 logs of concentration, a tight correlation was established between IC50 for the blockade of both processes, Na+/H+ exchange and percentage of cells entering the S phase upon growth factor action. These findings indicate that, in HCO-3-free medium, the functioning of the Na+/H+ exchange system is required for growth factor-induced DNA synthesis.

Amiloride↗
PubMed1984

A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH.

A H+-suicide technique based on the reversibility of Na+/H+ antiport was developed for the selection of mutants deficient in this membrane-bound activity. The strategy was to use the Na+/H+ antiporter as a H+-vector killing device. Chinese hamster lung fibroblasts (CCL39) were loaded with LiCl and incubated in Na+-, Li+-free choline Cl saline solution (pH 5.5). Under these conditions, intracellular pH dropped in 5 min from 7.1 to 4.8, leading to a rapid loss of cell viability (less than 0.1% survival after 30 min). Cytoplasmic acidification and cell death were prevented by treatment with 5-N,N-dimethylamiloride, a potent inhibitor of Na+/H+ antiport. Of the H+-suicide resistant clones that survived two cycles of selection, 90% were found deficient in Na+/H+ antiport activity. One class of mutants (PS10, PS12) fully resistant to the H+-suicide test, does not acidify the cell interior in response to an outward-directed Li+ gradient and has no detectable amiloride-sensitive Na+ influx measured either in Li+- or H+-loaded cells. Growth of these fibroblast clones lacking Na+/H+ antiport was found to be pH conditional in HCO3(-)-free medium. Whereas wild-type cells can grow over a wide range of external pHs (6.6-8.2), PS mutants cannot grow at neutral and acidic pHs (pH less than 7.2); their optimal growth occurs at alkaline pH values (pH 8-8.3). These findings strongly suggest that the Na+/H+ antiport activity through regulation of intracellular pH plays a crucial role in growth control.

Acid-Base Equilibrium↗
PubMed1984

Cytogenetic findings in acute promyelocytic leukaemia. A report of 25 cases.

Cytogenetic analyses of bone marrow cells were carried out by means of direct technique and short-term cultures in 25 acute promyelocytic leukaemia (APL) patients. 16 were affected by the hypergranular form and 9 by the M3-variant. The t(15;17) was documented at diagnosis in 15 patients. In addition, 2 cases, in which an M3 morphology and a normal karyotype on direct preparation were found at diagnosis, disclosed the chromosome anomaly in cultured cells at relapse, in concomitance with a change of blast morphology to the M3-variant. However, the t(15;17) was a consistent feature in 5 of the 15 patients with cells analysed on direct preparations only and in all cases who had successful bone marrow cultures. Overall, the 15;17 translocation was detected in 7 out of 9 patients with the M3-variant and in 8 out of 16 patients with typical M3. Whenever the chromosome preparations were made after culture, 7/7 had the translocation in the M3-V group and 5/5 in the M3 group. No obvious differences in clinical features or outcome were evident in the patients, irrespective of their cytogenetic findings.

Adolescent↗
PubMed1983

Growth factor-stimulated protein phosphorylation in G0/G1-arrested fibroblasts. Two distinct classes of growth factors with potentiating effects.

Protein phosphorylation of G0/G1-arrested Chinese hamster lung fibroblasts (CC139 line) has been analyzed following stimulation by fetal calf serum (FCS) or by a variety of growth factors. FCS stimulated the phosphorylation of three major polypeptides separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis: a nuclear protein with a Mr of 62,000 daltons, the ribosomal protein S6, and a cytosoluble peptide of 27,000 daltons. These phosphorylations occurred rapidly after serum stimulation (1 min for the 27,000-dalton peptide, 5 min for S6 and the 62,000-dalton proteins) and were maximal after 30 min. In nonstimulated cells the 27,000-dalton phosphopeptide exists in two forms with isoelectric points of 5.7 and 6.0; serum increased the amount of the most acidic form. At low concentrations, the "commitment" growth factors, alpha-thrombin, eye-derived growth factor (EDGF), platelet-derived growth factor (PDGF), stimulated phosphorylation of the 27,000-dalton peptide. At higher concentrations, these factors alone reinitiated DNA synthesis and, like FCS, stimulated phosphorylation of the three major peptides. In contrast, and suggesting a different mechanism of action, "progression" factors such as insulin (1-10 micrograms/ml) and multiplication-stimulating activity (MSA) are unable to stimulate phosphorylation of the 27,000-dalton peptide. However, insulin or MSA which are known to potentiate the mitogenic action of alpha-thrombin, PDGF, EDGF, ... were also found to potentiate phosphorylation of the ribosomal protein S6. These results support the existence of two classes of growth factors and suggest that protein phosphorylation is an early event involved in the control of the cellular G0 leads to G1 transition.

Animals↗
PubMed1983

Analysis of growth factor "relaxation" in Chinese hamster lung fibroblasts required for tumoral expression.

The Chinese hamster lung fibroblast line, CCl39, displays the properties characteristic of normal secondary cultures of Chinese hamster fibroblasts including: reversible G0 growth arrest (less than 2% labeled nuclei), anchorage dependence, and high serum-growth factor dependence. Injection of CCl39 cells, or anchorage-independent variants, in nude mice leads to tumor formation; however, as we have previously shown (Pérez-Rodriguez et al., 1981b), the resulting tumor clones no longer possess the high serum dependence of injected CCl39 cells. Hormonal growth restraints imposed by the host create an in vivo selection for diminished, or "relaxed," growth factor requirement. To characterize this growth factor "relaxation" further, we have analyzed the mitogenic response of parental CCl39 cells, anchorage-independent clones, and selected tumoral derivatives, to purified growth factors. Two highly purified growth factors, thrombin and insulin, together fulfill the growth factor requirements of CCl39 cells; thrombin (1 U/ml) stimulates the reinitiation of DNA synthesis in G0-arrested CCl39 cells, and insulin (10 micrograms/ml) maximally potentiates this stimulation to the level obtained with 10% fetal calf serum. First, we found no correlation between loss of anchorage dependence and growth factor relaxation. Second, we found that A71 (anchorage independent), a tumoral variant of CCl39 capable of growth arrest, and tumor-derived cells all display an increased sensitivity to thrombin and a diminished requirement for the potentiating action of insulin. Examination of thrombin binding to CCl39, A51 (nontumoral, anchorage independent), and A71 cells revealed that the increased sensitivity to thrombin of A71 cells is not attributable to an alteration in thrombin cell surface receptor number or affinity for thrombin. Rather, under standard conditions of serum or growth factor removal (30 hr), A71 cells maintain a metabolically elevated growth-arrested state, different from that of their nontumoral counterparts. Consequently, much lower concentrations of growth factors are needed to induce a proliferative response in these tumoral cells.

Animals↗
PubMed1982

Evidence that hamster fibroblasts tumors emerge in nude mice through the process of two in vivo selections leading to growth factor "relaxation" and to immune resistance.

The Chinese hamster lung fibroblast cell line (CC139), anchorage- and highly serum-dependent for growth is tumorigenic in nude mice. Tumors arise after 4 to 8 weeks following the inoculation of 5 X 10(5) cells. We have shown that all the emerging tumoral clones (more than 20 analyzed) have lost the growth factor dependence of the parental cells (Pérez-Rodriguez et al., 1981 a). To mimic this selection which occurred in vivo, we selected in vitro growth-factor-independent variants. These variants, GFI304 and GFI461, can proliferate in a serum-free medium supplemented with transferrin alone. This character is stable since it is not lost after the GFI variants have been cultivated in non-selective medium (serum) for more than 20 generations. In spite of their "autonomous" growth and anchorage independence. GFI variants are poorly tumorigenic. The observation of nodule formation and subsequent regression in nude mice, immunosuppressed (irradiation or cyclophosphamide) or not, led us to the conclusion that at least two selections in vivo are required for the tumoral expression of CC139 cells. One leads to a loss of growth factor requirement, the second towards a resistance to the immune surveillance mechanisms of the nude mice. The 4- to 8-week lag period of tumor formation may be accounted for by the spontaneous emergence in vivo of the two new characters necessary to bypass host growth restraints. This report supports the concept of stepwise progression and clonal evolution of preneoplastic cells in vivo and also indicates that tumorigenicity tests in nude mice should be interpreted with caution.

Animals↗
PubMed1982

Down-regulation of the hexose transport system: metabolic basis studied with a fibroblast mutant lacking phosphoglucose isomerase.

Down-regulation ("curb") of hexose transport in Chinese hamster lung fibroblasts has been studied in a metabolic mutant highly defective in phosphoglucose isomerase (PGI; glucosephosphate isomerase; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9). In the parental strain (PGI+) glucose as well as glucosamine and mannose were able to elicit a curb of the hexose transport system. In the PGI mutant, only glucose was able to mediate a transport curb. The inability of glucosamine and mannose to promote a transport curb in the PGI strain must be ascribed to the fact that the 6-esters of these aldohexoses are converted by their own specific deaminase and isomerase to fructose 6-phosphate, which initiates the pyruvate-tricarboxylate energy-yielding pathway but cannot be converted to glucose 6-phosphate in the mutant. The latter ester can be metabolized, but its metabolism in the mutant is confined to the pentose shunt. It is shown that inhibitors such as 2,4-dinitrophenol and malonate exert only slight inhibition of the pentose shunt yet release the glucose-mediated curb elicited by glucose and glucosamine in the parental PGI+ strain and also the glucose transport curb persisting in the PGI mutant.

Animals↗
PubMed1982

Growth factor activation of an amiloride-sensitive Na+/H+ exchange system in quiescent fibroblasts: coupling to ribosomal protein S6 phosphorylation.

Chinese hamster lung fibroblast cells (CCl39) enter the G0/G1 nonproliferative state after serum deprivation. In this report, we show that reinitiation of DNA synthesis by serum or the combination of purified human thrombin and insulin (1-10 microgram/ml) is preceded by very early stimulation of ionic fluxes (Na+/Rb+) and protein phosphorylation (27,000 daltons, 62,000 daltons, and the ribosomal S6 proteins). The potentiating action of insulin on thrombin-stimulated DNA synthesis is also observed on thrombin-stimulated Na+ influx, Rb+ influx, and protein S6 phosphorylation. Moreover, we demonstrate that CCl39 cells possess a Na+/H+ exchange system sensitive to amiloride. Half-maximal inhibition of growth factor-activated Na+ influx and Na+-dependent H+ efflux is obtained with 3-10 microM amiloride. Two lines of evidence indicate that the extrusion of H+ via the activation of the Na+/H+ exchanger is coupled to protein S6 phosphorylation: serum-stimulated phosphorylation is blocked by (i) amiloride at a concentration that abolishes serum-stimulated Na+ influx and (ii) protonophores that acidify the cell interior. The present data support the idea that the regulation of intracellular pH is a key event in the mechanism of growth factor action.

Amiloride↗
PubMed1981

Emergence of hamster fibroblast tumors in nude mice--evidence for in vivo selection leading to loss of growth factor requirement.

The Chinese hamster lung fibroblast cell line (CC139) has high anchorage dependence for growth and has retained the high serum dependence of secondary cultures of adult fibroblasts. This cell line is tumorigenic in nude mice; however, the resulting tumor cells have different properties than those of the cell line injected. The tumor-derived cells had strongly reduced or even lost both the high anchorage and the high serum dependence of CC139 cells. This finding suggests that an in vivo selection is necessary for CC139 cells to acquire the malignant phenotype. After mutagenesis, which increases the frequency of CC139 colony formation in agarose up to 8-fold, we selected and analyzed 15 anchorage-independent colonies. No correlation between the colony-forming ability in agarose and serum-growth factor requirement for DNA synthesis was observed. Each of these clones were injected into nude mice and the growth factor dependence of the ensuing tumor cells was compared to that of corresponding injected cells. All of the anchorage-independent colonies with the exception of one (A71), had acquired in vivo a stable phenotype allowing for partial or total escape of growth factor requirement. A71, the only clone which maintained the same growth factor requirement after two passages in vivo (A71 T1 and A71 T2) had already gained, in vitro, the minimal growth factor "relaxation" compatible with in vivo growth. A71 and A71 T1 tumor cells arrested in G0/G1 can reinitiate DNA synthesis in the presence of mouse plasma, low concentrations of serum, or thrombin. The fact that none of the tumors analyzed (more than 20) were found to have retained the high serum dependence of CC139 cells strongly suggests that the partial loss of serum growth factor requirement acquired in vivo is an essential malignant character for bypassing the hormonal growth restraints imposed by the host upon CC139 cells.

Animals↗
PubMed1981

Growth factor requirements of Chinese hamster lung fibroblasts in serum free media: high mitogenic action of thrombin.

We have defined a chemical medium to grow an anchorage and highly serum-dependent Chinese Hamster Lung fibroblast cell line (CCl 39) in the absence of serum. This serum-free medium referred to as DM4, contains Transferrin (5 microgram/ml), Insulin (10 microgram/ml), Epidermal Growth Factor (10 ng/ml) and Thrombin (0.05 U/ml). Thrombin appeared to be a very potent mitogen for Chinese Hamster Lung cells. DM4 allows exponential growth of CCl 39 cells plated in serum-coated dishes without any lag and with a generation time of 15 hours. Continuous subculturing and clonal growth can be obtained by passaging the cells with Ca++, Mg++ free phosphate-buffered saline in fibronectin coated dishes. The entire definition of growth factor requirements for this anchorage dependent cell line is of great value to approach the mechanisms of escape of growth control by these cells.

Animals↗
PubMed1980

Relationship between increased aerobic glycolysis and DNA synthesis initiation studied using glycolytic mutant fibroblasts.

Reports from several laboratories have suggested increased rates of glycolysis play an essential part in the initiation of DNA synthesis. This is based on observations that aerobic glycolysis: (1) occurs at low rate in resting mammalian cells and at very high rate in tumour cells; (2) increases rapidly after DNA synthesis is initiated by addition of serum or purified growth factors, and (3) correlates with the expression of the transformed phenotype. Also, specific inhibitors of aerobic glycolysis prevent the initiation of DNA synthesis. To determine whether the rapid activation of phosphofructokinase--and therefore glycolysis--by purified growth factors is necessary for the initiation of cell proliferation, we have isolated and studied two classes of glycolytic mutants. The first, isolated from Chinese hamster fibroblasts, has a total block in the glycolytic pathway. The second, from hamster and Fisher rat fibroblasts maintains a permanent high rate of glycolysis. We have found that both classes of mutants retain normal control of DNA synthesis in response to serum. This dissociation indicates that growth-factor-stimulated glycolysis is not involved in the control of initiation of DNA synthesis and cell proliferation.

Aerobiosis↗
PubMed1980

Isolation of a Chinese hamster fibroblast mutant defective in hexose transport and aerobic glycolysis: its use to dissect the malignant phenotype.

A procedure is described for the selection of glucose uptake mutants based upon radiation suicide of Chinese hamster fibroblasts by 2-deoxy[3H]glucose. In one of these mutants, DS 7, the ability to transport either 2-deoxyglucose or 3-O-methylglucose was decreased to one-fifth to one-fourth. Besides this defect, DS7 produces 1/14th the lactic acid produced by the parent when grown on 5 mM glucose. This block in aerobic glycolysis is due to a mutation that affects the expression of the phosphoglucose isomerase gene because no isomerase activity is detected in cell extracts of DS7. This glycolytic block makes that cell line dependent exclusively on respiration for its energy requirement. Consequently, DS7 survives well after removal of glucose but dies quickly in the presence of oligomycin. The parental line O23 (subclone of CCl39) grows at low serum concentration, is anchorage-independent, and is tumorigenic in nude mice. The derived glycolytic mutant DS7 has retained both the in vitro transformed phenotype (low serum dependence and loss of anchorage dependence) and the tumor-forming capability. The tumor cells derived from the injection of DS7 cells have kept the original glycolytic defect. This finding suggests that the transformed properties (high hexose transport and aerobic glycolysis) that can be uncoupled from abnormal growth control are not necessary for the expression of the malignant phenotype in fibroblasts.

Animals↗
PubMed1980

Cell mutants as a tool to study malignant transformation of fibroblasts.

From 3T3 Balb/c cells, mutants with low adhesiveness to plastic substratum were selected. One of these clones, AD6, was found to be deficient in glucosamine-6-phosphate N-acetylase. A result of this block is a decrease by 60-70% of cell surface carbohydrates, and as a consequence there is a general reduction in the exposure of glycoproteins at the cell surface. This biochemical defect is fully reverted to normal, simply by growing the mutant cells in presence of 10 mM N-acetylglucosamine. This specific and reversible enzymatic block allows us to conclude that the abnormal properties of AD6 cells--low adhesion, round shape, increased agglutinability by lectins, loss of directional locomotion, and absence of microfilament bundles--are the result of the surface carbohydrate defect, since reversion of glycoprotein synthesis to normal results in the general reversion of the altered phenotype. However, in spite of this apparent transformed phenotype, AD6 cells have normal growth control and are not tumorigenic. Using [3H]-2-deoxyglucose suicide, we selected from a spontaneously transformed clone of Chinese hamster lung fibroblasts a mutant (DS7) impaired in glucose metabolism, DS7 has a fourfold to fivefold decreased ability to transport either 2-deoxyglucose or 3-O-methylglucose and produces 14 times less lactic acid than the wild-type when grown on 5 mM glucose. This block in aerobic glycolysis, which is located at the level of phosphoglucose isomerase, makes that cell line dependent exclusively on respiration for its energy requirement. The parenteral line grows at low serum concentration, is anchorage-independent, and is tumorigenic in nude mice. The derived DS7 cells have retained both the in vitro transformed phenotype and the tumor-forming capability. A general conclusion of these studies is that the altered properties and cell behavior of transformed fibroblasts, such as low adhesiveness, round morphology, increased agglutinability by lectins, altered motility, absence of microfilament bundles, and increase in hexose transport and in aerobic glycolysis, are dissociable from malignant transformation. These phenotypic alterations should therefore be considered as events secondary to the mechanism that leads to the loss of growth control.

Animals↗
PubMed1980

[The therapy with synthetic antimalarial drugs controlled by fluorescence electroretinography (author's transl)].

Chloroquine retinopathy is a drug disease that can be prevented. Among some suggested methods, fluorescence electroretinography allows an evaluation of drug retinal deposit. In fact if a fluorescent substance accumulates in the retinal cells, a very short wavelength stimulus can produce a bioelectric response. If soon detected drug absorption is reversible. Only in a long run, the drug can induce irreversible damages. Thus the importance of continuous control of the patients in treatment with chloroquine-drugs is necessary. In this study the results of an investigation of 38 patients treated with synthetic anit-malarial drugs are shown. In some subjects the presence of the drug in the retina, without anatomic lesions, was demonstrated. These deposits disappeared upon cessation of the treatment. Deposits with or without retinal lesions were noticed in 31% of the observed subjects. In these patients the suspension of drug administration reduced accumulation but did not influence retinal lesion shown by fluorangiography. The authors hypothesized that fluorescent substance deposit is made easier by retinal vasculopathy due to connective tissue disorders.

Chloroquine↗