Relevance of drug uptake,cellular distribution and cell membrane fluidity to the enhanced sensitivity of Down's syndrome fibroblasts to anticancer antibiotic-mitoxantrone

Sensitivity of human fibroblasts derived from Down's syndrome (DS) individuals (S-240, T-158, T-74, T-164) and normal donors (S-126, WA-1) to anticancer antibiotic-mitoxantrone (1,4-dihydroxy-5,8-bis((2-((2-hydroxy-ethyl)amino)ethyl)amino)-9,10-anthracenedione dihydrochloride; MIT) and its relationship to the transport rate, cellular distribution and interaction with cell membrane were studied. The survival assay showed that MIT was more toxic to trisomic fibroblast lines than to normal cells. Studies of transport kinetics indicated that the amount of drug taken up and extruded by DS cells was diminished, compared to control cells. In contrast, the cellular level of MIT associated with DNA was greater in trisomic than in normal cells. The fluorescence anisotropy measurements of TMA-DPH and 12-AS demonstrated that the fluidity of the polar region of the outer lipid monolayer of DS cell membrane was decreased in comparison with normal cells. MIT treatment decreased fluidity of the inner hydrophobic region of plasma membrane, but only slightly influenced the fluidity of the outer surface of the cell membrane. Finally, we concluded that lowered membrane fluidity, diminished amount of MIT extruded by cells and the enhanced level of the drug associated with DNA could be responsible for the enhanced sensitivity of DS fibroblasts to the MIT treatment.     

Involvement of glutathione and glutathione-related enzymes in the protection of normal and trisomic human fibroblasts against daunorubicin

We measured the glutathione content, and the activity of glutathione-related enzymes and DT-diaphorase in cultured normal (cell line: S-126) and trisomic (cell lines: S-158, S-240) human fibroblasts exposed to daunorubicin (DNR). Determination of reduced and total glutathione levels, and measurement of the activity of glutathione peroxidase, glutathione reductase, glutathione-S-transferase and DT-diaphorase were performed spectrophotometrically. Human fibroblasts were exposed to 4 microm DNR for 2 h, and the cells placed in drug-free medium for 6, 12, 24, 48, and 72 h. Cellular levels of GSH and total glutathione decreased following exposure to DNR. However, the ratio of GSH to total glutathione returned to control levels only in trisomic cells. These changes were concomitant with increasing glutathione-S-transferase and glutathione reductase activities. DNR also significantly increased the activity of Se-independent peroxidase and DT-diaphorase in trisomic fibroblasts. Marked increases in the activity of Se-dependent peroxidase and DT-diaphorase alone were seen in normal cells. The results provide the first evidence that DNR can induce alterations in the level of glutathione and glutathione-dependent enzymes in trisomic fibroblasts as compared to normal cells, which may provide additional protection against daunorubicin-induced oxidative stress in trisomic fibroblasts.     

The induction of apoptosis by daunorubicin and idarubicin in human trisomic and diabetic fibroblasts

In this study, we investigated apoptosis induced in human trisomic and diabetic fibroblasts by daunorubicin (DNR) and its derivative, idarubicin (IDA). The cells were incubated with DNR or IDA for 2 h and then cultured in a drug-free medium for a further 2–48 h. The apoptosis in the cultured cell lines was assessed by biochemical analysis. We found that both drugs induced a timedependent loss of mitochondrial membrane potential, and a significant increase in intracellular calcium and caspase-3 activity. Mitochondrial polarization and changes in the level of intracellular calcium were observed during the first 2–6 h after drug treatment. Caspase-3 activation occurred in the late stages of the apoptotic pathway. Our findings also demonstrated that idarubicin was more cytotoxic and more effective than daunorubicin in inducing apoptosis in trisomic and diabetic fibroblasts.     

Contact-mediated changes in the fluidity of membrane lipids in normal and malignant transformed mammalian fibroblasts

The degree of microviscosity, gh, (fluidity/rigidity behavior) of membrane lipids of normal and transformed mammalian fibroblasts obtained from mice, hamsters and rats was quantitatively monitored by fluorescence polarization, P, analysis of the fluorescent probe 1,6-diphenyl 1,3,5-hexatriene (DPH) when embedded in lipid regions of cellular membranes of intact viable cells. Analysis of membrane microviscosity of six different cell populations and of individual cells in each cell population have indicated that the membrane microviscosity of all cell types, both normal and transformed fibroblasts, changes as a function of the cell density in the growing cultures. The membrane microviscosity was found to be low (high lipid fluidity) in sparse conditions but high (high lipid rigidity) in dense conditions. The induced changes in membrane microviscosity are practically reversible for all cell types and a complete reversion can be obtained within a few hours after changing the cell density conditions from sparse to dense and vice versa.Comparative studies with normal and transformed fibroblasts have shown that transformed fibroblasts have a more rigid lipid layer in their cellular membranes than normal or untransformed fibroblasts. The difference in membrane microviscosity between transformed and normal fibroblasts is higher in confluent conditions as compared with subconfluent cultures. These differences in the degree of fluidity of membrane lipids that are controlled by possible differences in the cellto-cell contact in normal and transformed fibroblasts may play a major role in determining the growth behavior of normal and malignant cells that are growing as a solid tissue and may have a direct effect on the control mechanisms that determine the presence or absence of the “density dependent inhibition” of growth.     

Analysis of aclarubicin-induced cell death in human fibroblasts

In the present study we investigated the mode of cell death induced by aclarubicin (ACL) in trisomic (BB) and normal (S-2) human fibroblasts. Cells were incubated with ACL for 2h and then cultured in drug-free medium for up to 96h. Using fluorescence microscopy, agarose gel electrophoresis and comet assay we demonstrate that ACL induced time-dependent morphological and biochemical changes in both cell types. The population of apoptotic cells, analysed by acridine orange and ethidium bromide nuclear staining reached its maximum at 24-48h. Prolonged post-treatment time progressively increased the level of necrotic cells. At 24-48h time points we also observed a significant increase in caspase-3 activity, oligonucleosomal DNA fragmentation and DNA strand breaks. Cotreatment of cells with the specific caspase-3 inhibitor Ac-DEVD-CHO partly reduced the extent of apoptosis and necrosis and DNA degradation. In conclusion, trisomic and normal fibroblasts demonstrate similar response to aclarubicin treatment. Drug induced the apoptotic and necrotic pathway of cell death that was mediated by caspase-3.     

Characteristics of intracellular metabolism of procollagen and other proteins in human embryo fibroblasts in trisomy for chromosome 7

A comparative study of the relative rates of intracellular total protein metabolism in diploid and aneuploid (with trisomy for chromosome 7) human embryo fibroblasts in the logarithmic and stationary growth phases was carried out. Using double labeling with [14C]proline (24 hrs) and [3H]proline (3 hrs), it was found that: the rates of intracellular protein metabolism during transition to the stationary phase of growth are increased in diploid cells and decreased in cells with trisomy for chromosome 7; the relative rate of protein metabolism in the logarithmic phase is higher in trisomic cells than in diploid ones. The intracellular degradation of procollagen in trisomic cells is increased approximately by 17% as compared to normal fibroblasts. Treatment of cell lysates with bacterial collagenase revealed the presence of procollagen incomplete degradation products in anomalous fibroblasts. The observed differences in the rates and mode of protein metabolism during transition of diploid and trisomic fibroblasts to the stationary phase of growth suggest that the odd autosome interferes with the normal coordinated activity of genes in chromosomes.     

Membrane perturbation through novel cell-penetrating peptides influences intracellular accumulation of imatinib mesylate in CML cells

Chronic myeloid leukemia is a stem cell disease with the presence of Philadelphia chromosome generated through reciprocal translocation of chromosome 9 and 22. The use of first- and second-generation tyrosine kinase inhibitors has been successful to an extent. However, resistance against such drugs is an emerging problem. Apart from several drug-resistant mechanisms, drug influx/efflux ratio appears to be one of the key determinants of therapeutic outcomes. In addition, intracellular accumulation of drug critically depends on cell membrane fluidity and lipid raft dynamics. Previously, we reported two novel cell-penetrating peptides (CPPs), namely, cationic IR15 and anionic SR11 present in tryptic digest of Abrus agglutinin. Here, the potential of IR15 and SR11 to influence intracellular concentration of imatinib has been evaluated. Fluorescent correlation spectroscopy and lifetime imaging were employed to map membrane fluidity and lipid raft distribution following peptide-drug co-administration. Results show that IR15 and SR11 are the two CPPs which can modulate membrane fluidity and lipid raft distribution in K562 cells. Both IR15 and SR11 significantly reduce the viability of CML cells in the presence of imatinib by increasing the intracellular accumulation of the drug.     

Membrane lipid dynamics and density dependent growth control in normal and transformed avian cells

Membrane fluidity of normal chick embryo fibroblasts and normal Japanese quail fibroblasts and their Rous sarcoma virus and methylcholanthrene transformed counterparts was investigated using the technique of fluorescence depolarisation of 1,6-diphenylhexatriene incorporated in the whole cells and in their isolated plasma membrane vesicles. Normal cells and isolated plasma membranes of normal cells showed significant changes in fluidity as a function of population density while neither Rous sarcoma virus transformed nor methylcholanthrene tumor cells or their isolated plasma membrane showed this effect. Stimulation of growth by addition of calf serum to cultures of quiescent, density-inhibited normal cells was accompanied by rapid changes in the direction of increased membrane lipid fluidity. Neither sparse normal cells, nor sparse or dense transformed cells showed any significant fluidity change in their membrane lipids upon addition of serum. Enzyme and electron microscopic analysis of the ratios of different membrane types in each cell type showed that this ratio was invariant with respect to cell population density but different between transformed and normal cells. Hence, the fluidity changes observed, measured as the mean rotational correlation time of the fluorescene probe in the membrane lipids, truly reflect organisational differences, occurring as a function of population density in cultures of cells which retain density-dependent growth control.     

Influence of daunorubicin on membrane permeability properties: detection by means of intracellular accumulation and efflux of fluorescein

The influence of daunorubicin (DNR) on membrane permeability properties was assessed by studying the ability of living HeLa cells to exhibit fluorochromasia; that is, to take up a fluorogenic substrate and retain the fluorescent compound obtained by enzymatic reaction. The intracellular accumulation of the fluorescent product as well as its release by the cells may be considered indicators of the permeability properties, since both processes are mediated by the cell membrane. The influence of the drug on the accumulation and on the efflux of fluorescein, obtained intracellularly from the hydrolysis of fluorescein diacetate (FDA), was evaluated, after DNR treatment, by measuring the fluorescence intensity of the product in single living cells by flow cytometry. The results showed that DNR, up to a concentration of 5 X 10(-6) M, did not significantly affect the accumulation of fluorescein. On the contrary, the efflux was strongly inhibited. A comparative study of the influence of drugs with known action mechanism was performed with the membrane-active compound hydrocortisone (HC) and with the metabolic inhibitor KCN. The results obtained indicate that DNR significantly affects membrane permeability properties and that its influence is similar to that exerted by metabolic inhibitors.     

Fluorescence-polarization measurements on normal and mutant human skin fibroblasts

Measurements of fluorescence polarization in intact diploid skin fibroblasts after exposure to 1,6-diphenyl-1,3,5-hexatriene were used to estimate the fluidity of the lipid phase(s) of cellular membranes. The membrane lipids of cells derived from four patients with homozygous familial hypercholesterolemia were in a more fluid state than those of cells obtained from 13 other individuals of normal and nonrelated mutant genotypes when all cultures were grown on medium with native serum. The only other cell type having membrane lipids of increased fluidity under these conditions was one fibroblast line derived from a patient with the Lesch-Nyhan syndrome. Examination of two additional nonconsanguinous lines of Lesch-Nyhan fibroblasts, however, revealed that an abnormally high level of lipid fluidity was not a common property of the membranes of cells of this genotype. Incubation of cultures in medium containing lipid-depleted serum (virtually devoid of lipoprotein-bound sterol) caused a reversible increase in the fluidity of the membranes of normal cells to values similar to those of the hypercholesterolemic cells, but had no effect on the membranelipid fluidity of the latter. By contrast, exposure of cultures to cholesterol not bound to lipoprotein in serum-free medium resulted in a decrease in the lipid fluidity of the membranes of both normo- and hypercholesterolemic fibroblasts.     

Improvement of flow-cytometric detection of multidrug-resistant cells by cell-volume normalization of intracellular daunorubicin content

To improve the ability of flow cytometry to detect multidrug-resistant cells, we studied the extent to which cell volume heterogeneity accounts for the variance of intracellular daunorubicin (DNR) content. For P388 murine or HL-60 human leukemia cells exposed to DNR (1 micrograms/ml, 60 min), log intracellular DNR content varied in direct proportion to log cell volume measured by flow cytometry, with a correlation coefficient of .9. This relationship was confirmed by cell sorting based on intracellular DNR content with subsequent volume determination of the sorted cells. Normalization of intracellular DNR content for cell volume (thus obtaining intracellular DNR concentration) was accomplished by subtracting log cell volume from log intracellular DNR content for each cell. This resulted in a 34% decrease (range 23-58%) in standard deviation compared to DNR content measurements without volume normalization for all cell types tested. Following exposure to DNR (as above), intracellular DNR content of drug-sensitive P388 or HL-60 cells measured by flow cytometry was 12- and 8-fold greater than that of the multidrug-resistant sublines P388/ADR and HL-60/AR, respectively. However, because of the variance of intracellular DNR content, the predictive value of flow-cytometric determination of intracellular DNR content as a discriminant assay for detecting the frequency of drug-resistant cells in a mixed population was acceptable only when the frequency of resistant cells in the population exceeded 10%. In contrast, volume normalization of intracellular DNR content enhanced the ability of the flow-cytometric assay to discriminate resistant cells by 10-fold for P388 cells and 100-fold for HL-60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in GSH-antioxidant system induced by daunorubicin in human normal and diabetic fibroblasts

We investigated the effect of daunorubicin on glutathione content and activity of GSH-related enzymes in cultured normal and diabetic human fibroblasts. Cells were incubated with 4 microM daunorubicin (DNR) for 2 h followed by culture in drug-free medium for up to 72 h. Treatment of diabetic cells with the drug caused a time-dependent depletion of intracellular GSH and a decrease of the GSH to total glutathione ratio. GSH depletion was accompanied by apoptotic changes in morphology of the nucleus. Analysis of GSH-related enzymes showed a significant increase of the activities of Se-dependent and Se-independent peroxidases and glutathione S-transferase. In contrast, glutathione reductase activity was reduced by 50%. Significant differences between normal and diabetic cells exposed to DNR were observed in the level of GST and Se-dependent glutathione peroxidase activities. These findings indicated that daunorubicin efficiently affects the GSH antioxidant defense system both in normal and diabetic fibroblasts leading to disturbances in glutathione content as well as in the activity of GSH-related enzymes.     

The influence of intracellular idarubicin and daunorubicin levels on drug cytotoxicity in childhood acute leukemia

Uptake and efflux of two anthracyclines, idarubicin (IDA) and daunorubicin (DNR), was studied in childhood acute leukemia samples. A comparison of IDA and DNR transport phenomena in relation to drug cytotoxicity and expression of P-glycoprotein (PGP) was made. Intracellular content of IDA/DNR was determined by flow cytometry using the fluorescent properties of the drugs. In vitro drug cytotoxicity was measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. PGP expression was analysed by flow cytometry. The uptake and efflux rates were non-significantly higher for IDA than DNR. There were no differences between three types of leukemia with respect to drug content during accumulation and retention. After correction for the cell volume, intracellular concentration of both drugs in each moment of uptake and efflux was significantly lower in relapsed ALL and AML samples in comparison with initial ALL cells. Efflux, but not uptake, of both drugs was inversely correlated with PGP expression and IDA, but not DNR, cytotoxicity. The cytotoxicity was correlated with drug accumulation for both drugs and with drug retention for IDA. In conclusion, it seems that (1) intracellular content was related to the lipophilic properties of the drugs rather than to the type of leukemia, (2) decreased intracellular concentration of both drugs might have an impact on compromised therapy results in AML and relapsed ALL children, (3) IDA presents higher cytotoxicity, which possibly might be decreased by the presence of PGP. These results might have a practical impact on the rational design of new chemotherapy protocols.     

Intracellular transport of cholesterol in type C Niemann-Pick fibroblasts

The purpose of this study was to determine the capacity of Niemann-Pick type C (NPC) fibroblasts to transport cholesterol from the cell surface to intracellular membranes. This is relevant in light of the observations that NPC cells display a sluggish metabolism of LDL-derived cholesterol, a phenomenon which could be explained by a defective intracellular transport of cholesterol. Treatment of NPC cells for 4 h with 0.1 mg/ml of LDL failed to increase the incorporation of [14C]oleic acid into cholesterol [14C]oleate, an observation consistent with previous reports on this cell type (Pentchev et al. (1985) Proc. Natl. Acad. Sci. USA 82, 8247). Normal fibroblasts, however, displayed the classical upregulation (6-fold over control) of the endogenous esterification reaction in response to LDL exposure. Incubation of normal or NPC fibroblasts with sphingomyelinase (100 mU/ml; Staphylococcus aureus) led to a rapid and marked increase (9- and 10-fold for normal and NPC fibroblasts, respectively, after 4 h) in the esterification of plasma-membrane-derived [3H]cholesterol suggesting that sphingomyelin degradation forced a net transfer of cholesterol from the cell surface to the endoplasmic reticulum. The similar response in normal and mutant fibroblasts to the degradation of sphingomyelin suggests that plasma membrane cholesterol can be transported into the substrate pool of ACAT to about the same extent in these two cell types. Degradation of cell sphingomyelin in NPC fibroblasts also resulted in the movement of 20-25% of the cellular cholesterol from a cholesterol oxidase susceptible pool into oxidase-resistant pools, implying that a substantial amount of plasma membrane cholesterol was internalized after sphingomyelin degradation. This cholesterol internalization was not accompanied by an increased rate of membrane internalization, as measured by [3H]sucrose uptake. Although NPC cells showed a relative accumulation of unesterified cholesterol and a sluggish esterification of LDL-derived cholesterol when exposed to LDL, these cells responded like normal fibroblasts with regard to their capacity to transport cholesterol from the cell surface into intracellular sites in response to sphingomyelin degradation. It therefore appears that NPC cells, in contrast to the impaired intracellular movement of lipoprotein-derived cholesterol, do not display a general impairment of cholesterol transport between the cell surface and the intracellular regulatory pool of cholesterol.     

Intracellular targeting of the insulin-regulatable glucose transporter (GLUT4) is isoform specific and independent of cell type

Insulin stimulates glucose transport in adipocytes via the rapid redistribution of the GLUT1 and GLUT4 glucose transporters from intracellular membrane compartments to the cell surface. Insulin sensitivity is dependent on the proper intracellular trafficking of the glucose transporters in the basal state. The bulk of insulin-sensitive transport in adipocytes appears to be due to the translocation of GLUT4, which is more efficiently sequestered inside the cell and is present in much greater abundance than GLUT1. The cell type and isoform specificity of GLUT4 intracellular targeting were investigated by examining the subcellular distribution of GLUT1 and GLUT4 in cell types that are refractory to the effect of insulin on glucose transport. Rat GLUT4 was expressed in 3T3-L1 fibroblasts and HepG2 hepatoma cells by DNA-mediated transfection. Transfected 3T3-L1 fibroblasts over-expressing human GLUT1 exhibited increased glucose transport, and laser confocal immunofluorescent imaging of GLUT1 in these cells indicated that the protein was concentrated in the plasma membrane. In contrast, 3T3-L1 fibroblasts expressing GLUT4 exhibited no increase in transport activity, and confocal imaging demonstrated that this protein was targeted almost exclusively to cytoplasmic compartments. 3T3-L1 fibroblasts expressing GLUT4 were unresponsive to insulin with respect to transport activity, and no change was observed in the subcellular distribution of the protein after insulin administration. Immunogold labeling of frozen ultrathin sections revealed that GLUT4 was concentrated in tubulo-vesicular elements of the trans-Golgi reticulum in these cells. Sucrose density gradient analysis of 3T3-L1 homogenates was consistent with the presence of GLUT1 and GLUT4 in discrete cytoplasmic compartments. Immunogold labeling of frozen thin sections of HepG2 cells indicated that endogenous GLUT1 was heavily concentrated in the plasma membrane. Sucrose density gradient analysis of homogenates of HepG2 cells expressing rat GLUT4 suggested that GLUT4 is targeted to an intracellular location in these cells. The density of the putative GLUT4-containing cytoplasmic membrane vesicles was very similar in HepG2 cells, 3T3-L1 fibroblasts, 3T3-L1 adipocytes, and rat adipocytes. These data indicate that the intracellular trafficking of GLUT4 is isoform specific. Additionally, these observations support the notion that GLUT4 is targeted to its proper intracellular locale even in cell types that do not exhibit insulin-responsive glucose transport, and suggest that the machinery that regulates the intracellular targeting of GLUT4 is distinct from the factors that regulate insulin-dependent recruitment to the cell surface.     

Sorting of an internalized plasma membrane lipid between recycling and degradative pathways in normal and Niemann-Pick, type A fibroblasts

We examined the metabolism and intracellular transport of a fluorescent sphingomyelin analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl])- sphingosylphosphorylcholine (C6-NBD-SM), in both normal and Niemann-Pick, type A (NP-A) human skin fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 3 min of warming to 37 degrees C, both normal and NP-A fibroblasts had internalized C6-NBD-SM from the plasma membrane, resulting in a punctate pattern of intracellular fluorescence. Rates for C6-NBD-SM internalization and transport from intracellular compartments to the plasma membrane (recycling) were similar for normal and NP-A cells. With increasing time at 37 degrees C, internalized C6-NBD-SM accumulated in the lysosomes of NP-A fibroblasts, while normal fibroblasts showed increasing Golgi apparatus fluorescence with no observable lysosomal labeling. Since NP-A fibroblasts lack lysosomal (acid) sphingomyelinase (A-SMase), this result suggested that hydrolysis of C6-NBD-SM prevented its accumulation in the lysosomes of normal fibroblasts during its transport along the degradative pathway. We used the amount of C6-NBD-SM hydrolysis by A-SMase in normal cells as a measure of C6-NBD-SM transported from the cell surface to the lysosomes. After a lag period, C6-NBD-SM was delivered to the lysosomes at a rate of approximately 8%/h. This rate was approximately 18-19 fold slower than the rate of C6-NBD-SM recycling from intracellular compartments to the plasma membrane. Thus, small amounts of C6-NBD-SM were transported along the degradative pathway, while most endocytosed C6-NBD-SM was sorted for transport along the plasma membrane recycling pathway.     

Role of membrane lipids and membrane fluidity in thermosensitivity and thermotolerance of mammalian cells

The role of membrane lipids and membrane fluidity in thermosensitivity of mammalian cells is not well understood. The limited experimental data in the literature have led to conflicting results. A detailed investigation of lipid composition and membrane fluidity of cellular membranes was undertaken to determine their relationship to cell survival after hyperthermia. Ehrlich ascites (EA) cells, mouse fibroblast LM cells, and HeLa S3 cells differed in thermosensitivity as expressed by a D0 of 3.1, 5.2, and 9.7 min, respectively, at 44 degrees C. No correlation with cellular thermosensitivity could be found with respect to the amount of cholesterol and to the cholesterol to phospholipid ratio in the particulate fraction of the cells. By growing the cells for some generations in different media, cholesterol and phospholipid content could be changed in the particulate fraction, but no difference in cell survival was observed. When mouse fibroblasts were grown for 24 hr in a serum-free medium supplemented with arachidonic acid (20:4), all subcellular membranes were about eight times richer in phospholipids containing polyunsaturated acyl (PUFA) chains and membrane fluidity was increased as measured by fluorescence polarization of diphenylhexatriene (DPH). The alterations resulted in a higher thermosensitivity. When mouse fibroblasts were made thermotolerant no change in cholesterol and phospholipid content could be found in the particulate fraction of the cells. The relative weights and the quality of the phospholipids as well as the fatty acid composition of the phospholipids appeared to be the same for normal and thermotolerant cells. Fluidity measurements in whole cells, isolated plasma membranes, and liposomes prepared from phospholipids extracted from the cells revealed no significant differences between normal and thermotolerant fibroblasts when assayed by fluorescence polarization (DPH) and electron spin resonance (5-nitroxystearate). It is concluded that the mechanism of thermal adaptation resulting in differences in lipid composition as reported in the literature differs from the mechanism of the acquisition of thermal tolerance. The lower heat sensitivity of thermotolerant cells, as initiated by a nonlethal triggering heat dose followed by an induction period at 37 degrees C, does not involve changes in lipid composition and membrane fluidity. However, a prompt and clear (also nonlethal) change in membrane fluidity by an increase in PUFA does result in an increased thermosensitivity, probably because of an indirect effect via the lipids in causing disfunctioning of proteins in the membrane and/or the cytoskeleton.     

Epithelial-Stromal Interactions in Human Breast Cancer: Effects on Adhesion,Plasma Membrane Fluidity and Migration Speed and Directness

Interactions occurring between malignant cells and the stromal microenvironment heavily influence tumor progression. We investigated whether this cross-talk affects some molecular and functional aspects specifically correlated with the invasive phenotype of breast tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration) by co-culturing mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer-associated fibroblasts, CAFs) in 2D or 3D (nodules) cultures. Confocal immunofluorescence analysis of the epithelial adhesion molecule E-cadherin on frozen nodule sections demonstrated that NFs and CAFs, respectively, induced or inhibited its expression in MCF-7 cells. An increase in the mesenchymal adhesion protein N-cadherin was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of cancer cells. CAFs, in turn, promoted N-cadherin up-regulation in MDA-MB-231 cells and its de novo expression in MCF-7 cells. Beyond promotion of “cadherin switching”, another sign of the CAF-triggered epithelial-mesenchymal transition (EMT) was the induction of vimentin expression in MCF-7 cells. Plasma membrane labeling of monolayer cultures with the fluorescent probe Laurdan showed an enhancement of the membrane fluidity in cancer cells co-cultured with NFs or CAFs. An increase in lipid packing density of fibroblast membranes was promoted by MCF-7 cells. Time-lapsed cell tracking analysis of mammary cancer cells co-cultured with NFs or CAFs revealed an enhancement of tumor cell migration velocity, even with a marked increase in the directness induced by CAFs.Our results demonstrate a reciprocal influence of mammary cancer and fibroblasts on various adhesiveness/invasiveness features. Notably, CAFs'' ability to promote EMT, reduction of cell adhesion, increase in membrane fluidity, and migration velocity and directness in mammary cancer cells can be viewed as an overall progression- and invasion-promoting effect.     

Plasmalogen content and beta-adrenoceptor signalling in fibroblasts from patients with Zellweger syndrome. Effects of hexadecylglycerol

In Zellweger or cerebro-hepato-renal syndrome (CHRS), the assembly of peroxisomes is defective, resulting in deficient plasmalogen formation. Plasmalogens are part of the membrane lipid composition. In fibroblasts of CHRS patients, the plasmalogen fraction of phosphatidylethanolamine (PPE) was about half of that in control cells while total phospholipid (PL) content, individual PL and plasma membrane fluidity were normal. CHRS cell strains had higher beta-adrenoceptor numbers and isoproterenol-stimulated cAMP responses. Receptors were more efficiently coupled to adenylate cyclase than in control cells. Stimulations of cAMP with NaF or forskolin were the same as in control cells. Restoring synthesis of plasmalogens with hexadecylglycerol (HDG), a plasmalogen precursor, resulted in a proportionate increase in PPE of about 40% in both control and CHRS fibroblasts. Exposure to HDG reduced surface beta-adrenoceptor sites and cAMP-responses to isoproterenol in CHRS cells only, while post-receptor stimulations of cAMP were reduced in both cell types. Plasmalogen contents inversely correlated with isoproterenol-stimulated cAMP levels. The increased numbers of functional beta-adrenoceptors in CHRS fibroblasts may be the result of a higher expression and/or of a prolonged functional half-life of the receptor protein. In vivo, this may contribute to the clinical manifestations of the disease.     

Progressive muscular dystrophy (Duchenne): Biochemical studies by flow-cytometry

Summary The peanut lectin (PNL) receptor density of the cell membrane and several metabolic parameters of cultured fibroblasts of normal human individuals and of patients with muscular dystrophy were measured by simultaneous two and three parameter flow cytometry. The PNL-receptor density was significantly decreased on muscular dystrophy fibroblasts (between 20.7 and 33.6%) as compared to normal fibroblasts. The cell volume, the esterase activity, the intracellular pH, and the percentage of proliferating cells of both types of fibroblasts were not significantly altered. The mean cell volume of different fibroblast cultures varied between 2500 and 6000m³. The concentration of the intracellular esterase activity of fibroblasts was low (0.169 relative units) as compared to lymphocytes and granulocytes of the peripheral blood (1.56 and 2.17 relative units). The fibroblasts had an acidic intracellular pH of 6.52 while lymphocytes and granulocytes had basic pH values of 7.30 and 7.17. Some of the fibroblasts were in the S+G2/M phase of the cell cycle (20%). The study shows that the measurement of biochemical parameters of vital and fixed single fibroblasts by flow-cytometry is of great interest for the recognition of differences between normal individuals and muscular dystrophy patients.