CV1-P Cells Research Articles

Cytotoxicity of curcumin, resveratrol and plant extracts from basil, juniper, laurel and parsley in SH-SY5Y and CV1-P cells

The health-promoting effects of natural phenolic compounds are attracting growing interest. In this study, we tested four plant extracts, namely basil, juniper, laurel and parsley, and two well-known pure phenolic compounds of natural origin, curcumin and resveratrol, for their possible cytotoxic effects in vitro. AraC was used as an apoptotic reference compound. The pure compounds were studied in two cell lines, SH-SY5Y neuroblastoma and CV1-P fibroblast cells, and the plant extracts in SH-SY5Y cells. Cytotoxicity was examined by MTT and LDH assays, and the molecular mechanisms by Western blot analysis of p53 protein in cells. Juniper extract decreased the cell viability and increased the amount of p53 in SH-SY5Y cells at lower concentrations than did other plant extracts, and its effects on the amount of p53 in cells were comparable to the treatment with 50 μM AraC. The actions of curcumin and resveratrol were dependent on the concentration and cell line. Curcumin decreased the cell viability and increased the amount of p53 in SH-SY5Y cells more effectively than in CV1-P cells. The comparison between the results of AraC and pure curcumin showed a similar effect on the amount of p53. Our results indicate a potential role of plant extracts and plant-derived compounds as health-promoting food constituents, as well as candidates for drug development.

Inhibition of Simian Virus 40 Large T Antigen Helicase Activity by Fluoroquinolones

Fluoroquinolones represent a potent group of antibiotics that inhibit bacterial DNA replication by targeting the essential bacterial enzymes gyrase and topoisomerase IV. Inhibition of gyrase activity by quinolones involves the interaction of these drugs with the helicase component of bacterial gyrase. DNA tumour viruses also encode helicases that are essential for their DNA replication in the host. In this study we have evaluated the effect of fluoroquinolones on viral DNA replication using the DNA tumour virus Simian virus 40 (SV40) as our model. Four different fluoroquinolones, namely, levofloxacin, trovafloxacin, ciprofloxacin and ofloxacin, were tested for their ability to inhibit viral DNA replication. We show here that all four quinolones tested were effective in the inhibition of SV40 plaque formation and DNA replication in CV1-P cells. In addition, we found that each of these quinolones was inhibitory to the helicase activity of SV40 large tumour antigen. Fluoroquinolones and their derivates may therefore be useful in the treatment and/or prevention of infection by SV40-homologous human DNA viruses that encode helicase activity for their survival.

A prolyl oligopeptidase inhibitor, Z-Pro-Prolinal, inhibits glyceraldehyde-3-phosphate dehydrogenase translocation and production of reactive oxygen species in CV1-P cells exposed to 6-hydroxydopamine

We studied the ability of prolyl oligopeptidase (POP) inhibitors, Z-Pro-Prolinal and JTP-4819, to prevent translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and formation of reactive oxygen species (ROS), in 6-hydroxydopamine (6-OHDA) and cytosine arabinoside (Ara-C) treated monkey fibroblast (CV1-P) and human neuroblastoma (SH-SY5Y) cells. The cells were pretreated with POP inhibitors (30 min) before addition of toxicants. GAPDH was analyzed by Western hybridization, ROS by fluorescent 2′7′-dichlorodihydro-fluorescein diacetate, and viability by the MTT method. Both toxicants induced GAPDH translocation to the particulate fraction (mitochondria and nuclei). Z-Pro-Prolinal was able to inhibit the translocation in 6-OHDA-exposed CV1-P cells. In SH-SY5Y cells and in JTP-4819 pretreated cells, no prevention of translocation was seen. However, the intensity of GAPDH in cytosolic fraction increased. Both inhibitors blocked 6-OHDA-induced ROS-production to the control level in CV1-P but, not in SH-SY5Y cells, without affecting their viability. In conclusion, POP inhibitors are able to prevent certain cell stress related factors such as ROS production or GAPDH translocation.

The roles of dopamine transporter and Bcl-2 protein in the protection of CV1-P cells from 6-OHDA-induced toxicity

6-Hydroxydopamine (6-OHDA) is widely used to produce an animal model of Parkinson’s disease by selectively destroying the catecholaminergic nerve system of the substantia nigra. In our previous studies we noted that dopaminergic neuroblastoma cells (SH-SY5Y) die mostly via apoptosis after exposure to 6-OHDA (≤100 μM) but African green monkey fibroblast (CV1-P) cells do not succumb, although in both cell lines there were increased intracellular p53 levels. This study was designed to further investigate the mechanisms underlying the p53 elevation. To test how 6-OHDA penetrates into fibroblast cells and affects p53 levels, we investigated the presence of the dopamine transporter (DAT) in CV1-P cells. We showed by western hybridization that CV1-P cells contain the DAT. The apparent entry of 6-OHDA into fibroblasts was decreased by the DAT inhibitor, 1-(2-bis-(4-fluorophenyl)methoxy)ethyl)-4-(3-phenyl-propyl)piperazine (GBR 12909). Pre-treatment with GBR 12909 decreased the elevation of intracellular ROS to the control level and thus prevented the increase of p53 levels in 6-OHDA-treated CV1-P cells. Moreover, an increase of Bcl-2, an antiapoptotic protein, was detected after 6-OHDA treatment, supporting our previous results where no increase in caspase-3 activity was detected. We suggest that Bcl-2 may block the activation of the caspase cascade and protect CV1-P cells from apoptosis.

The serine/threonine kinase cyclin G-associated kinase regulates epidermal growth factor receptor signaling.

Cyclin G-associated kinase (GAK) is a serine/threonine kinase that features high homology outside its kinase domain with auxilin. Like auxilin, GAK has been shown to be a cofactor for uncoating clathrin vesicles in vitro. We investigated epidermal growth factor (EGF) receptor-mediated signaling in cells, in which GAK is down-regulated by small hairpin RNAs. Here, we report that down-regulation of GAK by small hairpin RNA has two pronounced effects on EGF receptor signaling: (i) the levels of receptor expression and tyrosine kinase activity go up by >50-fold; and (ii) the spectrum of downstream signaling is significantly changed. One very obvious result is a large increase in the levels of activated extracellular signal-regulated kinase 5 and Akt. These two effects of GAK down-regulation result from, at least in part, alterations in receptor trafficking, the most striking of which is the persistence of EGF receptor in altered cellular compartment along with activated extracellular signal-regulated kinase 5. The alterations resulting from GAK down-regulation can have distinctive biological consequences: In CV1P cells, down-regulation of GAK results in outgrowth of cells in soft agar, raising the possibility that loss of GAK function may promote tumorigenesis.

Growth Inhibition of Macrophage-like and Other Cell Types by Liposome-encapsulated, Calcium-bound, and Free Bisphosphonates In Vitro

Bisphosphonates effectively inhibit osteoclastic bone resorption in diseases characterized by excessive bone loss. Liposome-encapsulated clodronate (dichloromethylene bisphosphonate) also is known to inactivate phagocytic cells in vivo, and inhibit the growth of macrophage-like Raw 264 cells in vitro. The macrophage suppressive effect of liposomal clodronate is of interest in autoimmune diseases, like rheumatoid arthritis, in which phagocytic cells are involved in inflammatory processes. Earlier in vivo studies suggested that liposomal clodronate is a far more potent inactivator of macrophages than liposomal forms of two other bisphosphonate compounds, pamidronate (3-amino-1-hydroxypropylidene bisphosphonate), and etidronate (1-hydroxyethylidene-1, 1-bisphosphonate). We examined the growth inhibitory properties of these three bisphosphonates with macrophage-like RAW 264 cells and with other types of cells in vitro. All three bisphosphonates encapsulated in liposomes effectively inhibited the growth of RAW 264 and CV1-P cells, while free drugs were 20–1000 times less potent growth inhibitors. Also, high extracellular calcium concentrations enhanced the potency of bisphosphonates for RAW 264 cells, indicating that, in addition to liposomes, the uptake of bisphosphonates by macrophages, is mediated also by calcium. In all formulations, pamidronate was the most potent compound for the cells, with the exception CV1-P cells, for which liposomal clodronate was the most potent. The effects of liposomal drugs were selective for highly endocytotic cells. The results suggest that liposome-encapsulated bisphosphonates could provide a specific tool to affect the function of macrophages and all three of these bisphosphonates are potentially effective as macrophage suppressors in autoimmune diseases.

Ara-C induces apoptosis in monkey fibroblast cells

The effect of cytosine arabinoside (Ara-C) on cell viability has been studied in African green monkey kidney fibroblasts (CV1-P). It has been shown previously that Ara-C- induced cell death in neurons is mediated by apoptosis. We investigated whether Ara-C can induce apoptosis also in CV1-P cells, and if the apoptosis is p53-associated. For comparison, human neuroblastoma cells (SH-SY5Y) were studied as a model of human neuronal cells. SYTO13/propidium iodide staining revealed condensed and fragmented nuclei in both cell lines. Ara-C treatment for 48 h induced ∼24% apoptosis in CV1-P cells whereas ∼55% of SH-SY5Y cells were apoptotic. Ara-C increased the level of p53 in both CV1-P and SH-SY5Y cells compared to control. The maximum level of p53 in SH-SY5Y cells was reached at 12 h and this then rapidly faded whereas CV1-P cells p53 levels remained elevated after reaching their maximum. Caspase-3 activity was 5-fold higher in human neuroblastoma cells than in monkey fibroblasts, this reflected the decreased cell viability. Our results prove that Ara-C- induced apoptosis in CV1-P cells is associated with an increase of p53 and activation of caspase-3. Ara-C-induced toxicity in CV1-P cells is modest compared to that seen in neuronal cells.

Hypoxia Stimulates p16 Expression and Association with cdk4

Exposure of CV-1P cells to hypoxic conditions causes cell proliferation inhibition concomitant with the accumulation of pRb in the hypophosphorylated, growth suppressive form. This is in part due to inhibition of pRb-directed cdk4 and cdk2 activity. In this study we attempted to elucidate the mechanism by which cdk4 is inactivated under hypoxic conditions. After 18 h of hypoxia, CV-1P cells are inhibited from progressing from G1 phase into the S phase of the cell cycle. This occurs in conjunction with dephosphorylation of serine-795, which is a putative substrate of cdk4. The amounts of cdk4, cdk6, and the D type cyclins are not affected by 18 h of hypoxia. The levels of cdki p16, p18, p19, and p57 under aerobic or hypoxic conditions were analyzed and although the levels of most cdki are unaffected by hypoxic conditions, the level of p16 increases significantly by 18 h of hypoxia. The mechanism by which cdk4 activity is inhibited under hypoxic conditions may be mediated through p16 association with cdk4. Immunoprecipitation analysis shows that p16 binds to cdk4 under hypoxic conditions but does not in cells maintained under aerobic conditions. Thus p16 may be involved in hypoxia-induced growth inhibition.

Cell Association of Liposomes with High Fluid Anionic Phospholipid Content is Mediated Specifically by LDL and Its Receptor, LDLr

We have sought to confirm indications in our recent studies suggesting that association of liposomes composed of 75–100 mol % egg phosphatidylglycerol (ePG), a fluid anionic phospholipid, with cells is mediated by low density lipoprotein (LDL) and the classical LDL receptor (LDLr). In the present study, binding of liposomes composed of 75–100 mol % ePG to CV1-P cells, either in serum-supplemented medium or in defined medium supplemented with LDL, is blocked by the presence of either of two monoclonal antibodies. The first is immunoglobulin (Ig)G C7, an antibody specific for LDLr. The second is IgG 5E11, an antibody specific for domain 3441-3569 of apolipoprotein B100. CHOldlA7, a cell line known to lack the LDLr and previously shown by us to associate minimally with 75–100 mol % ePG liposomes, was transfected with the human LDLr. The transfected cells bound 75–100 mol % ePG liposomes at high levels, and this binding was blocked by IgG C7. Previously, we have shown that serum, but not LDL or high density lipoprotein, induces association of 25–50 mol % ePG liposomes with both CV1-P and CHO wild type cells, but not CHOldlA7. In the present study, IgG C7 does not block this interaction, and transfected CHOldlA7 cells do not show this interaction. Hence, this form of liposome binding appears not to involve LDL or LDLr, but requires a receptor, currently unknown, and a serum component other than LDL or high density lipoprotein. The unknown receptor, in addition to LDLr, is missing from CHOldlA7. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:1233–1244, 2002

Extracellular and intracellular factors influencing gene transfection mediated by 1,4-dihydropyridine amphiphiles

Double-charged 1,4-dihydropyridine (1,4-DHP) amphiphiles have been shown to condense DNA and efficiently transfect it into cells in vitro [Hyvönen et al., Biochim. Biophys. Acta 1509 (2000) 451]. Alkyl chain length and buffering capacity at endosomal pH range (5.0–7.4) affected complexation and transfection activity. In this study we examined how those chemical modifications of amphiphile–DNA complexes (amphiplexes) affect their interactions with extracellular polyanions (glycosaminoglycans, albumin) and lipid bilayers, their cellular uptake and intracellular distribution. To evaluate cellular uptake, CV1-P cells were incubated with labeled DNA–amphiphile complexes and analyzed by flow cytometry. Confocal laser fluorescence microscopy was used to investigate the intracellular distribution of amphiplexes. The results showed that biophysical properties of compounds can be changed by slight structural modifications. These factors determine the intracellular kinetics and transfection efficacy of the compounds. Some extracellular glycosaminoglycans and serum interfere with 1,4-DHP-amphiphile-mediated transfection by destabilizing the amphiplexes. Neither high cellular uptake, membrane destabilizing activity nor buffering capacity alone is adequate for high transfection efficacy. The activity results from complex interplay of various factors that determine intracellular kinetics and, consequently, transfection.

The use of low-molecular-weight PEIs as gene carriers in the monkey fibroblastoma and rabbit smooth muscle cell cultures.

Polyethylenimines (PEIs) and cationic polymers have been used successfully in gene delivery. In earlier reports, only large PEIs (MW>10 000) have shown significant transfection efficiency. In the present study, the roles of small PEIs (MW 700 and 2000) were studied as additional compounds to see if they can improve gene delivery with cationic liposomes. The TKBPVlacZ expression plasmid was transfected in the CV1-P (monkey fibroblastoma) and SMC (rabbit smooth muscle) cell lines using various combinations of PEIs (MW 700, 2000, and 25 000) and Dosper liposomes. The transfection efficiency was determined with the fluorometric ONPG (o-nitrophenol-beta-D-galactopyranoside) assay and histochemical X-gal staining. The toxicity of the transfection reagents was estimated by the MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide] assay. Transfection of TKBPVlacZ plasmid by the small PEIs (MW 700 and 2000) combined with Dosper liposomes was associated with high expression of the lacZ reporter gene in the CV1-P and SMC cell lines. The transfection efficiencies of the low-molecular-weight PEI/liposome combinations were several fold higher than those of PEIs or liposomes alone. PEI/liposome combinations had no toxicity on the cell lines tested. The low-molecular-weight PEIs could be used successfully for gene delivery when combined with the cationic liposomes, resulting in a synergistic increase of the transfection efficiency in both cell lines studied.

Efficient Transfection of Novel Bovine Papillomavirus 1 Expression Plasmids

A series of novel bovine papillomavirus type 1 (BPV-1)-based expression plasmids was constructed and characterized in vitro as a starting point for the development of an in vivo gene therapeutic method. The order of transfection efficiency for different pBPVlacZ plasmids was pCGalBPV > pTKBPV > pSRαBPV in CV1-P cells. In the absence of selection pressure, the expression of pCGalBPVlacZ and pTKBPVlacZ was associated with long-term maintenance. In a comparison of pBPVlacZ with pSVlacZ, expression was maintained up to 12–17 and 8–12 days, respectively. The transfection of pBPVlacZ plasmids was efficient in secondary and primary, dividing and nondividing, neural and nonneural, and human cells and, furthermore, independent of the cell cycle as seen in growing as well as resting cells. All these characteristics are likely to be relevant for in vivo conditions, under which the percentage of proliferating cells could be quite low. In conclusion, the pBPV plasmids were efficiently delivered and expressed in different host cells, and therefore their performance in gene therapy is worth testing.

LDL-induced association of anionic liposomes with cells and delivery of contents: II. Interaction of liposomes with cells in serum-containing medium

In the present study we have investigated cell binding and drug delivery potency of various anionic liposomal formulations in a serum-supplemented growth medium, in order to understand the role of the LDL receptor in targeted drug delivery mediated by anionic liposomes. The cell lines used were CV1-P and CHO wild type, which both express the LDL receptor, and CHOldlA7, which lacks the LDL receptor. Cellular association of encapsulated methotrexate and fluorescein labeled phosphatidylethanolamine in the lipid bilayer was measured. Potency of two liposome-dependent drugs ( N-phosphonacetyl- l-aspartic acid and fluoroorotic acid) was also measured by growth inhibition. Association of ePG liposomal aqueous contents with cells grown in serum-supplemented growth medium was up to 30-fold higher with CV1-P or CHO wild type cells than with CHOldlA7. Increased association was not paralleled by a corresponding increase in potency of liposome-dependent drugs. The serum-dependent association of fluid, anionic (ePG) liposomes with cells expressing the LDL receptor is caused by an interaction of ePG liposomes with LDL. The failure of this association to increase drug delivery seems to be caused by the downregulation of LDL receptor expression when cells are continuously exposed to LDL.

LDL induced association of anionic liposomes with cells and delivery of contents as shown by the increase in potency of liposome dependent drugs.

To establish whether anionic liposomes interact with the low-density lipoprotein (LDL) receptor, to determine the role of lipoproteins in this interaction, and whether the association causes functional delivery of encapsulated drugs. The cell lines used were CV1-P and CHO wild type, both of which express the LDL receptor, and CHOldlA7, which lacks the LDL receptor. Cellular association of encapsulated methotrexate and fluorescein, labeled phosphatidylethanolamine in the lipid bilayer, was measured. Potency of three liposome dependent drugs (N-phosphonacetyl-L-aspartic acid, fluoroorotic acid, and methotrexate-gamma-aspartate) was also measured by growth inhibition. Association of liposomes containing at least 75 mol egg phosphatidylglycerol (ePG)/100 mol phospholipid with cells grown in defined medium supplemented with 1.0 mg/ml LDL was up to 30-fold higher with CV1-P or CHO wild type cells than with CHOldlA7, and 5-fold higher than association in defined medium lacking LDL. The addition of LDL did not yield any elevation of cellular association of distearoylphosphatidylglycerol liposomes. Increased association was paralleled by a corresponding increase in potency of all three liposome dependent drugs tested. ePG liposomes interact with the LDL receptor in an LDL-dependent fashion, and the interaction results in the delivery of contents to cells.

Hypoxia-induced pRB hypophosphorylation results from downregulation of CDK and upregulation of PP1 activities.

Exposure of CV-1P cells to hypoxic conditions results in reversible cell cycle arrest concomitant with accumulation of pRB in the hypophosphorylated, growth suppressive form. Similar to cell cycle arrest induced by serum starvation, we show here that hypoxia-induced arrest is accompanied by a decrease in pRB-directed CDK4 and CDK2 activities, lower cyclin D and E protein levels, and by an increase in p27 protein abundance. Immunoprecipitation studies reveal an increase in p27 association with cyclin E-CDK2 complexes. In contrast to cell cycle arrest induced by serum starvation, hypoxia increases PP1-mediated pRB dephosphorylation. These data reveal that synergy between decreased pRB-directed cyclin/CDK activity and increased pRB-directed phosphatase activity contribute towards inducing and maintaining pRB in its hypophosphorylated, growth suppressive state during hypoxia.

Transfection of BPV-LAC Z plasmids in primate CV1-P and SH-SY5Y cell cultures

Transfection of BPV-LAC Z plasmids in primate CV1-P and SH-SY5Y cell cultures

High Molecular Weight Protein Phosphatase Type 1 Dephosphorylates the Retinoblastoma Protein

pRb controls cell proliferation by restricting inappropriate entry of cells into the cell division cycle. As dephosphorylation of pRb during mitotic exit activates its growth suppressive function, identification of the protein phosphatase that dephosphorylates pRb, and characterization of the mechanism of its regulation, are essential to elucidating the mechanisms of cell growth control. By fractionating mitotic CV-1P cell extracts, we identify the protein phosphatase which dephosphorylates pRb as a type 1 serine/threonine phosphoprotein phosphatase (PP1). Molecular sizing analyses indicate that the catalytic enzyme (PP1c) is present in a high molecular weight complex, with a predicted molecular mass of 166 kDa. PP1-interacting proteins in the mitotic cell extracts are identified. Two PP1-interacting proteins (41 and 110 kDa) are shown to form distinct complexes with PP1c from fractions of separated mitotic cell extracts containing phosphorylase phosphatase activity. However, only the 110-kDa PP1-interacting protein is present in fractions containing pRb-directed phosphatase activity, identifying this protein as a putative activator of PP1 function toward pRb during mitosis.

Transrepression of RNA polymerase II promoters by the simian virus 40 small t antigen.

Simian virus 40 (SV40) small t antigen (t) can activate transcription from certain RNA polymerase II and III promoters (M. Loeken, I. Bikel, D. M. Livingston, and J. Brady, Cell 55:1171-1177, 1988). Here we report a new function of t, its ability to repress human c-fos promoter and AP-1 transcriptional activity in CV-1P cells. This function is the product of a discrete N-terminal domain of t, because the large T antigen (T)/t-common polypeptide, which contains only the first 82 amino acids common to both T and t of SV40, was, like the intact protein, an active repressor. The data further suggest that the t- and T/t-common-mediated repression of c-fos expression was most likely manifest at the level of transcription. In keeping with the possibility that t affects the expression of the genomic c-fos promoter, it also led to repression of AP-1 formation. Thus, SV40 is both an activator and a repressor of transcription. Its ability to inhibit c-fos expression should be considered in light of the natural history of SV40 in its natural host.

The kinetics of simian virus 40-induced progression of quiescent cells into S phase depend on four independent functions of large T antigen

Microinjection of purified simian virus 40 large-T-antigen protein or DNA encoding T antigen into serum-starved cells stimulates them to re-enter the cell cycle and progress through G1 into the S phase. Genetic analysis of T antigen indicated that neither its Rb/p107-binding activity nor its p53-binding activity is essential to induce DNA synthesis in CV1P cells. However, T antigens bearing missense mutations that inactivate either activity induced slower progression of the cells into the S phase than did wild-type T antigen. Inactivation of both activities resulted in a T antigen essentially unable to induce DNA synthesis. Missense mutations in either the DNA-binding region of the N terminus also impaired the ability of full-length T antigen to stimulate DNA synthesis in CV1P cells. The wild-type kinetics of cell cycle progression were restored by genetic complementation after coinjection of plasmid DNAs encoding different mutant T antigens or coinjection of purified mutant T-antigen proteins, suggesting that the four mitogenic functions of T antigen are independent. The maximal rate of induction of DNA synthesis in secondary primate cells and established rodent cell lines required the same four functions of T antigen. A model to explain how four independent activities could cooperate to stimulate cell cycle progression is presented.

Liposome-mediated delivery of gallium to macrophage-like cells in vitro: demonstration of a transferrin-independent route for intracellular delivery of metal ions.

Gallium (Ga) prevents the activation of macrophages and might be useful as an immunosuppressive agent. It is taken up by the malignant cells through the transferrin (Tf) receptor pathway, but this pathway may be insufficient in the case of non-malignant cells. We studied the Tf-independent, liposome-mediated delivery of Ga to macrophage-like cells in vitro by a growth inhibition assay. The growth inhibitory properties of Ga for other types of cells was also evaluated. Ga complexed with nitrilotriacetate (GaNTA) and encapsulated in DSPG-liposomes was 16 and 48 times more potent for RAW 264 cells than free GaNTA and Ga-nitrate, respectively. CV1-P cells were also somewhat sensitive to liposomal Ga, but other cell lines with lower endocytotic capacity were insensitive. The inhibition of RAW 264 cell growth induced by liposomal or free GaNTA was partially reversed with iron-loading of the cells, indicating that this form of Ga causes an intracellular iron deficiency similar to that produced by Tf-bound Ga. Our results indicate that encapsulation of Ga in negatively charged liposomes provides a transferrin independent route for intracellular delivery of the compound to macrophages, which is of special interest in the treatment of autoimmune diseases, such as rheumatoid arthritis.