Cellular Protein Secretion Research Articles

Differential Response of Mono Mac 6, BEAS-2B, and Jurkat Cells to Indoor Dust

BackgroundAirway toxicity of indoor dust is not sufficiently understood.ObjectivesOur goal in this study was to describe the effects of indoor dust on human monocyte, epithelial, and lymphocyte cell lines. We aimed to a) obtain a comprehensive and intelligible outline of the transcriptional response; b) correlate differential transcription with cellular protein secretion; c) identify cell line–specific features; and d) search for indoor dust–specific responses.MethodsSettled dust was sampled in 42 German households, and various contaminants were characterized. We exposed Mono Mac 6, BEAS-2B, and Jurkat cells to 500 μg/mL indoor dust for 6 hr. Outcome parameters included the transcriptional profile of an oligonucleotide microarray covering 1,232 genes. Significantly enriched Gene Ontology themes were calculated. Supernatant protein levels of 24 inflammatory response proteins served to confirm transcriptional results.ResultsAn intraclass correlation coefficient of 0.8 indicated reasonable microarray reproducibility. The transcriptional profile was characterized by enhancement of detoxification and a danger and defense response. Differential gene regulation correlated with protein secretion (Goodman and Kruskal’s gamma coefficient: 0.72; p < 0.01). Mono Mac 6 cells revealed the highest fraction of differentially expressed genes, dominated by up-regulation of various cytokines and chemokines. BEAS-2B cells revealed weaker changes in a limited set of inflammatory response proteins. No significant changes were observed in Jurkat cells.ConclusionsMonocytes are particularly responsive to indoor dust. We observed a classical T-helper 1-dominated immune response, which suggested that bioorganic contaminants are relevant effectors in indoor dust.

Landmark discoveries in intracellular transport and secretion

Cellular protein transport and secretion is fundamental to the very existence of an organism, regulating important physiological functions such as reproduction, digestion, energy production, growth, neurotransmission, hormone release, water and ion transport, etc., all required for the survival and maintenance of homeostasis within an organism. Molecular understanding of transport and secretion of intracellular product has therefore been of paramount importance and aggressively investigated for over six decades. Only in the last 20 years, the general molecular mechanism of the process has come to light, following discovery of key proteins involved in ER-Golgi transport, and discovery of the ‘porosome’– the universal secretion machinery in cells.

Lessening Hydroxyapatite Ceramics Particles Adverse Effects by Serum Modification: A Preliminary In Vitro Study and Mechanism Analysis

Serum immersion, a new method of pre-treated HA ceramics, has been compared with SBF immersion and untreated HA particles in cell culture condition. MTT assay indicates that serum-immersion lessens particles cytotoxity of HA particles in cultured L929 mouse fibroblast cell (p&lt;0.05). Neither immersion nor traditional physical factors such as particle size, shape should be responsible for these because SBF immersion with the similar factors results in no significant improvement. The reason might be serum proteins adsorption and biommetic microstructure formation on the ceramics surface, which affect cellular membrane behaviors, phagosome metabolization and cellular protein secretion. Otherwise, cells with particles degraded early apparently, which indicated that particles influence the cell cycle. The whole processes must be correlated intensively with cytokine, protease and intracellular ion concentration, which would be discussed in our follow-up studies.

Genetically Encoded but Nonpolypeptide Prolyl-tRNA Functions in the A Site for SecM-Mediated Ribosomal Stall

The arrest sequence, FXXXXWIXXXXGIRAGP, of E. coli SecM interacts with the ribosomal exit tunnel, thereby interfering with translation elongation. Here, we studied this elongation arrest in vitro using purified translation components. While a simplest scenario would be that elongation is arrested beyond Pro166, the last arrest-essential amino acid, and that the Pro166 codon is positioned at the P site of the ribosomal peptidyl transferase center (PTC), our toeprint analyses revealed that the ribosome actually stalls when the Pro166 codon is positioned at the A site. Northern hybridization identification of the polypeptide bound tRNA and mass determination showed that the last amino acid of the arrested peptidyl-tRNA is Gly165, which is only inefficiently transferred to Pro166. Also, puromycin does not effectively release the arrested peptidyl-tRNA under the conditions of A site occupancy by Pro166-tRNA. These results reveal that secM-encoded Pro166-tRNA functions as a nonpolypeptide element in fulfilling SecM's role as a secretion monitor.

Inhibition of cellular protein secretion by picornaviral 3A proteins

During poliovirus infection, anterograde traffic between the endoplasmic reticulum and the Golgi is inhibited due to the action of 3A, an 87 amino acid viral protein. The ability of poliovirus protein 3A to inhibit ER-to-Golgi traffic is not required for virus growth. Instead, we have suggested that the inhibition of host protein secretion, shown to reduce the secretion of interferon-β, IL-6, and IL-8 and the expression of both newly synthesized MHC class I and TNF receptor in the plasma membrane of infected cells, affects growth in host organisms. To determine whether the ability of poliovirus 3A to inhibit ER-to-Golgi traffic is conserved, the ability of 3A proteins from several picornaviruses, including human rhinovirus 14, foot-and-mouth disease virus, enterovirus 71, hepatitis A, and Theiler's virus, was tested. Only the 3A proteins from another poliovirus, Sabin 3, and closely related coxsackievirus B3 inhibited ER-to-Golgi traffic as effectively as the 3A protein from poliovirus Mahoney type 1. Site-directed mutagenesis based on these findings and the three-dimensional structure of the amino-terminal domain of poliovirus 3A protein revealed that residues in the unstructured amino terminus of 3A are critical for the inhibition of host protein secretion.

Cryopreservation of alginate-encapsulated recombinant cells for antiangiogenic therapy.

The potential benefit of continuous local administration of antiangiogenic proteins to CNS tumors in vivo has recently been demonstrated using endostatin-producing recombinant cells encapsulated in alginate beads. Due to the treatment potential of transplanted alginate-encapsulated cells producing therapeutic proteins, we describe a successful method of cryopreservation (CP) of such beads, in which cellular viability, alginate structure, and protein secretion were maintained. Alginate beads containing human embryonic kidney cells (HEK 293 cells) stably transfected with the gene encoding for endostatin were cryopreserved in dimethyl sulfoxide (DMSO) using a slow freezing procedure. Briefly, the DMSO concentration was gradually increased prior to the freezing procedure. The cryotubes were further supercooled to -7.5 degrees C and nucleated. Thereafter, the samples were cooled at a rate of 0.25 degrees C/min and stored in liquid nitrogen. The viability of the encapsulated cells was assessed using confocal microscopy quantification (CLSM) technique and a MTS assay. The cell cycle distribution inside the beads was assessed by DNA flow cytometry and endostatin production was determined by an endostatin-specific ELISA assay, both prior to and after CP. CLSM measurements showed sustained esterase activity in the beads after thawing, with only a slight transient decrease 24 h after CP. The MTS assay verified these findings by displaying similar variations of intracellular dehydrogenase activity. Flow cytometric analyses revealed no cryorelated disturbances in cellular ploidy. Furthermore, ELISA measurements showed a well-preserved endostatin production after CP. In conclusion, this work describes the successful CP of alginate-encapsulated recombinant cells secreting a therapeutic protein. Together with previous published reports, these results further substantiate the feasibility and potential of cell encapsulation therapy in the treatment of malignant tumors.

Production of a human calcitonin precursor with Staphylococcus carnosus: secretory expression and single-step recovery by expanded bed adsorption

Recombinant human calcitonin (hCT) can be expressed by Staphylococcus carnosus as a precursor peptide being part of a secreted fusion protein which contains the propeptide of a Staphylococcus hyicus lipase as secretion carrier and multiple repeats of the hCT precursor. Fusion proteins containing two (PhCT2), four (PhCT4), or eight (PhCT8) hCT precursors were expressed. Due to the unfavourable biochemical properties of PhCT4 and PhCT8, PhCT2 was the most efficient fusion protein for a production process. Expression from a xylose-inducible promoter from Staphylococcus xylosus resulted in a maximum secretory production of PhCT2, since the amount of PhCT2 synthesized in total corresponded almost precisely to the upper limit of the capacity of the cellular protein secretion apparatus. An expanded bed adsorption procedure for the recovery of PhCT2 from unclarified culture broth was established using the cation exchanger STREAMLINE SP at pH 5. This integrated operation allows a single-step recovery of PhCT2 and was successfully transferred to a pilot scale (100 l culture broth).

Ready…set…fuse

![][1] Fusion-competence is infectious. The fusion of two lipid membranes underlies a huge range of biological phenomena, from infection by enveloped viruses to the secretion of cellular proteins, but a central aspect of membrane fusion has remained mysterious: do the proteins that

Poliovirus 3A protein limits interleukin-6 (IL-6), IL-8, and beta interferon secretion during viral infection.

During viral infections, the host secretory pathway is crucial for both innate and acquired immune responses. For example, the export of most proinflammatory and antiviral cytokines, which recruit lymphocytes and initiate antiviral defenses, requires traffic through the host secretory pathway. To investigate potential effects of the known inhibition of cellular protein secretion during poliovirus infection on pathogenesis, cytokine secretion from cells infected with wild-type virus and with 3A-2, a mutant virus carrying an insertion in viral protein 3A which renders the virus defective in the inhibition of protein secretion, was tested. We show here that cells infected with 3A-2 mutant virus secrete greater amounts of cytokines interleukin-6 (IL-6), IL-8, and beta interferon than cells infected with wild-type poliovirus. Increased cytokine secretion from the mutant-infected cells can be attributed to the reduced inhibition of host protein secretion, because no significant differences between 3A-2- and wild-type-infected cells were observed in the inhibition of viral growth, host cell translation, or the ability of wild-type- or 3A-2-infected cells to support the transcriptional induction of beta interferon mRNA. We surmise that the wild-type function of 3A in inhibiting ER-to-Golgi traffic is not required for viral replication in tissue culture but, by altering the amount of secreted cytokines, could have substantial effects on pathogenesis within an infected host. The global inhibition of protein secretion by poliovirus may reflect a general mechanism by which pathogens that do not require a functional protein secretory apparatus can reduce the native immune response and inflammation associated with infection.

Evidence of Rab3A expression, regulation of vesicle trafficking, and cellular secretion in response to heregulin in mammary epithelial cells.

Heregulin beta1 (HRG), a combinatorial ligand for human growth factor receptors 3 and 4, is a regulatory polypeptide that promotes the differentiation of mammary epithelial cells into secretory lobuloalveoli. Emerging evidence suggests that the processes of secretory pathways, such as biogenesis and trafficking of vesicles in neurons and adipose cells, are regulated by the Rab family of low-molecular-weight GTPases. In this study, we identified Rab3A as a gene product induced by HRG. Full-length Rab3A was cloned from a mammary gland cDNA library. We demonstrated that HRG stimulation of human breast cancer cells and normal breast epithelial cells induces the expression of Rab3A protein and mRNA in a cycloheximide-independent manner. HRG-mediated induction of Rab3A expression was blocked by an inhibitor of phosphatidylinositol 3-kinase but not by inhibitors of mitogen-activated protein kinases p38(MAPK) and p42/44(MAPK). Human breast epithelial cells also express other components of regulated vesicular traffic, such as rabphilin 3A, Doc2, and syntaxin. Rab3A was predominantly localized in the cytosol, and HRG stimulation of the epithelial cells also raised the level of membrane-bound Rab3A. HRG treatment induced a profound alteration in the cell morphology in which cells displayed neuron-like membrane extensions that contained Rab3A-coated, vesicle-like structures. In addition, HRG also promoted the secretion of cellular proteins from the mammary epithelial cells. The ability of HRG to modify exocytosis was verified by using a growth hormone transient-transfection system. Analysis of mouse mammary gland development revealed the expression of Rab3A in mammary epithelial cells. Furthermore, expression of the HRG transgene in Harderian tumors in mice also enhanced the expression of Rab3A. These observations provide new evidence of the existence of a Rab3A pathway in mammary epithelial cells and suggest that it may play a role in vesicle trafficking and secretion of proteins from epithelial cells in response to stimulation by the HRG expressed within the mammary mesenchyma.

Aβ peptides and calcium influence secretion of the amyloid protein precursor from chick sympathetic neurons in culture

The major constituent of amyloid plaques in the Alzheimer disease (AD) brain is the amyloid protein (Aβ). Aβ has been shown to be neurotoxic to cells, but the exact mechanism of its effects are still not known. Most studies have focussed on Aβ neurotoxicity, but little is known about the effect of Aβ peptides on cellular protein metabolism and secretion. To examine the effect of Aβ peptides on APP secretion, chick sympathetic neurons were metabolically labeled with [35S]methionine and the amounts of radiolabeled APP and Aβ quantitated. Several Aβ peptides (Aβ25–35, [pyroglu3]Aβ3–40, and [pyroglu11]Aβ11–40) inhibited secretion of [35S]APP and increased cell-associated [35S]APP. There was also a 2–2.5-fold increase in secretion of several other proteins when cells were incubated with Aβ25–35. However, the amount of Aβ secreted into the medium was decreased. Treatment of cells with the calcium ionophore A23187 caused a 1.5-fold increase in secreted [35S]APP and a decrease in cell-associated [35S]APP. Although L-type voltage-dependent calcium channels (VDCC) have been implicated in Aβ toxicity, the effect of L-type VDCC on APP secretion has not previously been examined. The L-type VDCC antagonists nifedipine and diltiazem both increased [35S]APP secretion into the medium but did not influence the effect of Aβ on [35S]APP secretion. These studies suggest that Aβ interferes with the secretory pathway of APP. Insofar as secreted APP has been proposed to have a neuroprotective function, the accumulation of Aβ in the AD brain could decrease secreted APP and thereby indirectly increase Aβ toxicity. J. Neurosci. Res. 61:449–457, 2000. © 2000 Wiley-Liss, Inc.

A beta peptides and calcium influence secretion of the amyloid protein precursor from chick sympathetic neurons in culture.

The major constituent of amyloid plaques in the Alzheimer disease (AD) brain is the amyloid protein (A beta). A beta has been shown to be neurotoxic to cells, but the exact mechanism of its effects are still not known. Most studies have focussed on A beta neurotoxicity, but little is known about the effect of A beta peptides on cellular protein metabolism and secretion. To examine the effect of A beta peptides on APP secretion, chick sympathetic neurons were metabolically labeled with [(35)S]methionine and the amounts of radiolabeled APP and A beta quantitated. Several A beta peptides (A beta(25-35), [pyroglu(3)]A beta(3-40), and [pyroglu(11)]A beta(11-40)) inhibited secretion of [(35)S]APP and increased cell-associated [(35)S]APP. There was also a 2-2.5-fold increase in secretion of several other proteins when cells were incubated with A beta(25-35). However, the amount of A beta secreted into the medium was decreased. Treatment of cells with the calcium ionophore A23187 caused a 1.5-fold increase in secreted [(35)S]APP and a decrease in cell-associated [(35)S]APP. Although L-type voltage-dependent calcium channels (VDCC) have been implicated in A beta toxicity, the effect of L-type VDCC on APP secretion has not previously been examined. The L-type VDCC antagonists nifedipine and diltiazem both increased [(35)S]APP secretion into the medium but did not influence the effect of A beta on [(35)S]APP secretion. These studies suggest that A beta interferes with the secretory pathway of APP. Insofar as secreted APP has been proposed to have a neuroprotective function, the accumulation of A beta in the AD brain could decrease secreted APP and thereby indirectly increase A beta toxicity.

Imino sugars inhibit the formation and secretion of bovine viral diarrhea virus, a pestivirus model of hepatitis C virus: implications for the development of broad spectrum anti-hepatitis virus agents.

One function of N-linked glycans is to assist in the folding of glycoproteins by mediating interactions of the lectin-like chaperone proteins calnexin and calreticulin with nascent glycoproteins. These interactions can be prevented by inhibitors of the alpha-glucosidases, such as N-butyl-deoxynojirimycin (NB-DNJ) and N-nonyl-DNJ (NN-DNJ), and this causes some proteins to be misfolded and retained within the endoplasmic reticulum (ER). We have shown previously that the NN-DNJ-induced misfolding of one of the hepatitis B virus (HBV) envelope glycoproteins prevents the formation and secretion of virus in vitro and that this inhibitor alters glycosylation and reduces the viral levels in an animal model of chronic HBV infection. This led us to investigate the effect of glucosidase inhibitors on another ER-budding virus, bovine viral diarrhea virus, a tissue culture surrogate of human hepatitis C virus (HCV). Here we show that in MDBK cells alpha-glucosidase inhibitors prevented the formation and secretion of infectious bovine viral diarrhea virus. Data also are presented showing that NN-DNJ, compared with NB-DNJ, exhibits a prolonged retention in liver in vivo. Because viral secretion is selectively hypersensitive to glucosidase inhibition relative to the secretion of cellular proteins, the possibility that glucosidase inhibitors could be used as broad-based antiviral hepatitis agents is discussed. A single drug against HBV, HCV, and, possibly, HDV, which together chronically infect more than 400 million people worldwide, would be of great therapeutic value.

Secretory function of the Fallopian tube epithelial cells in vitro: A review

The oviduct provides a specialized environment for the survival and transport of the mammalian gametes to the site of fertilization within well defined time limits. The oviduct environment facilitates the process of fertilization and development of the preembryo. Furthermore, the spermatozoa and the pre-embryo, which differ antigenically from the mother, are not attacked by the immune system, which differentiates them from pathogens. The mechanisms by which all these complex processes are controlled are not fully understood. The disadvantage of cell culture systems for physiological study is that the original histological appearance is lost along with some endocrine and paracrine activities. Cells cultured on glass or plastic surfaces lose the polarization normally present in intact tissues. Premature senescence and de-differentiation may occur. It has been shown that extracellular matrix may stimulate proliferation and prevent dedifferentiation. The use of polarized cell cultures also enable the study of cellular orientation and protein secretion. Culture systems for polarized and non-polarized Fallopian tube epithelial cells have been developed in our laboratory. The subsequent experimental studies have shown that the presence of oviduct epithelial cells has a specific and significant stimulatory effect on sperm capacitation, reversing effects on the blockage of blastocyst formation. The exact mechanism of how the co-culture system achieves this is yet to be elucidated and in this regard, the unique oviduct proteins are possible candidates. Further studies of the epithelial cell cultures directed at the nature of the secretory behavior of these cells, are necessary in order to determine how these cells behave in vitro .

Interleukin 1beta mediates the modulatory effects of monocytes on LNCaP human prostate cancer cells.

Proliferative and secretory responses in androgen-sensitive prostate cancer LNCaP cells are regulated by steroid and peptide hormones and by differentiation-promoting substances. In the present study, we evaluated whether peripheral blood monocytes that exhibit anti-tumour activity in haematopoietic and solid tumours influence growth and secretion in the LNCaP cell line. For this purpose, LNCaP cells were incubated with monocyte-conditioned medium (MCM), and proliferation as well as expression of androgen receptor (AR) and secretion of prostate-specific antigen (PSA) were assessed. Conditioned medium from monocytes reduced proliferation in a dose-dependent manner. Incubation with 40% MCM caused a 50% reduction in cell proliferation. AR protein decreased by 70% and PSA levels in supernatants from LNCaP cells were reduced by approximately 80% following treatment with MCM. We focused on the contribution of two major products of activated monocytes, prostaglandin E2 and interleukin 1beta (IL-1beta), to the MCM modulatory action. LNCaP cells treated with prostaglandin E2 showed neither a reduction in proliferation nor a down-regulation of AR and PSA levels. The effects of MCM on cellular proliferation, AR protein and PSA secretion were abolished by pretreatment of MCM with a neutralizing anti-IL-1beta antibody. In addition, recombinant IL-1beta was able to replace MCM for the inhibition of proliferation and down-regulation of AR and PSA proteins. LNCaP cells were shown to express the IL-1beta receptor type 1, which transduces IL-1beta signal. Our findings reveal that monocyte-derived IL-1beta inhibits the proliferation of androgen-responsive prostate tumour cells and reduces AR and PSA levels.

Coexpression of molecular chaperone BiP improves immunoglobulin solubility and IgG secretion from Trichoplusia ni insect cells.

Infection of Trichoplusia ni (BTI-TN5B1-4) insect cells with a baculovirus coding for immunoglobulin G resulted in significant intracellular insolubility of the immunoglobulin chains. In order to increase the immunoglobulin solubility, the chaperone BiP was coexpressed in the insect cells using a separate baculovirus vector. This heterologous BiP was observed to associate with immunoglobulin chains in vivo and enhance the level of soluble intracellular and secreted IgG obtained from T.ni. Pulse chase studies indicated that the heterologous BiP increased the level of soluble nascent immunoglobulin chains and assembly intermediates to suggest that BiP is acting as a true molecular chaperone. The effect of heterologous BiP became more significant with time post-infection as secreted IgG levels increased by 90% after 3.4 days of baculovirus infection. Even following the treatment of cells with tunicamycin, BiP coexpression still enhanced immunoglobulin solubility and secretion to indicate that BiP does not function specifically to retain unglycosylated proteins in the endoplasmic reticulum. Thus, coexpression of a molecular chaperone may be used to enhance cellular productivity and protein secretion provided that the chaperone is involved with post-translational processing and significant protein aggregation is observed.

Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A.

Poliovirus RNA replication occurs on the surface of membranous vesicles that proliferate throughout the cytoplasm of the infected cell. Since at least some of these vesicles are thought to originate within the secretory pathway of the host cell, we examined the effect of poliovirus infection on protein transport through the secretory pathway. We found that transport of both plasma membrane and secretory proteins was inhibited by poliovirus infection early in the infectious cycle. Transport inhibition did not require viral RNA replication or the inhibition of host cell translation by poliovirus. The viral proteins 2B and 3A were each sufficient to inhibit transport in the absence of viral infection. The intracellular localization of a secreted protein in the presence of 3A with the endoplasmic reticulum suggested that 3A directly blocks transport from the endoplasmic reticulum to the Golgi apparatus.

Neutrophil chemotaxis in response to TGF-beta isoforms (TGF-beta 1, TGF-beta 2, TGF-beta 3) is mediated by fibronectin.

TGF-beta isoforms regulate numerous cellular functions including cell growth and differentiation, the cellular synthesis and secretion of extracellular matrix proteins, such as fibronectin (Fn), and the immune response. We have previously shown that TGF-beta 1 is the most potent chemoattractant described for human peripheral blood neutrophils (PMNs), suggesting that TGF-beta s may play a role in the recruitment of PMNs during the initial phase of the inflammatory response. In our current studies, we demonstrate that the maximal chemotactic response was attained near 40 fM for all mammalian TGF-beta isoforms. However, there was a statistically significant difference in migratory distance of the PMNs: TGF-beta 2 (556 microM) > TGF-beta 3 (463 microM) > TGF-beta 1 (380 microM) (beta 2: beta 3, p < or = 0.010; beta 3: beta 1, p < or = 0.04; beta 2: beta 1, p < or = 0.0012). A mAb to the cell binding domain (CBD) of Fn inhibited the chemotactic response to TGF-beta 1 and TGF-beta 3 by 63% and to TGF-beta 2 by 70%, whereas the response to FMLP, a classic chemoattractant, was only inhibited by 18%. In contrast, a mAb to a C-terminal epitope of Fn did not retard migration (< 1.5%). The Arg-gly-Asp-ser tetrapeptide inhibited chemotaxis by approximately the same extent as the anti-CBD (52 to 83%). Furthermore, a mAb against the VLA-5 integrin (VLA-5; Fn receptor) also inhibited TGF-beta-induced chemotaxis. These results indicate that chemotaxis of PMNs in response to TGF-beta isoforms is mediated by the interaction of the Arg-gly-Asp-ser sequence in the CBD of Fn with an integrin on the PMN cell surface, primarily the VLA-5 integrin. TGF-beta isoforms also elicited the release of cellular Fn from PMNs; we observed a 2.3-fold increase in Fn (389 to 401 ng/ml) in the supernatants of TGF-beta-stimulated PMNs compared with unstimulated cells (173.6 ng/ml). The concentration of TGF-beta required to cause maximal release of Fn from PMNs (4000 fM) is a concentration at which TGF-beta is no longer chemotactic, suggesting that PMNs only use Fn that is constitutively expressed for migration. At higher concentrations of TGF-beta, the Fn released may accumulate basal to the cell, ultimately retarding cellular migration and modulating the chemotactic response.

Peptide transport by the multidrug resistance pump.

The membrane P-glycoprotein (P170) is an ATP-hydrolyzing transmembrane pump, and elevated levels of P170, due to higher expression with or without amplification of the multidrug resistance gene (mdr1), result in resistance to a variety of chemotherapeutic agents in mammalian cells. The function of the P170 pump has been proposed as a protection against toxic substances present in animal diets. Here we describe a Chinese hamster ovary cell line that was selected for resistance to a synthetic tripeptide, N-acetyl-leucyl-leucyl-norleucinal (ALLN). This ALLN-resistant variant shows the classical multidrug resistance (MDR) phenotype, including overexpression and amplification of the mdr1 gene. Additionally, a mouse embryo cell line overexpressing the transfected mdr1 gene is likewise resistant to ALLN. Our results demonstrate that P170 is capable of transporting peptides and raise the possibility that the mdr1 gene product or other MDR-like genes, present in the genome of mammalian cells, may be involved in secretion of peptides or cellular proteins as is the case with the structurally similar hylB and ste6 gene products of Escherichia coli and yeast, respectively.

Inhibition of very-low-density lipoprotein secretion by chloroquine, verapamil and monensin takes place in the Golgi complex

The effects of chloroquine, verapamil and monensin on secretion of very-low-density lipoproteins (VLDLs) were studied in cultured rat hepatocytes. Maximum inhibition of VLDL-triacylglycerol secretion by 50–90% of control was reached at 200 μM chloroquine, 200 μM verapamil and 5 μM monensin, whereas no effect on cellular triacylglycerol synthesis was observed. The inhibition could be seen within 15 min and was reversible after washout of the drugs. Chloroquine and verapamil inhibited both cellular protein synthesis and protein secretion, whereas monesin reduced protein secretion without any effect on protein synthesis. Control experiments with cycloheximide revealed that intact protein synthesis was not necessary for secretion of VLDL-triacylglycerol during 2 h. Electron micrographs of cells treated with chloroquine, verapamil or monensin showed swollen Golgi cisternae containing VLDL-like particles. By morphometry, a more than 2-fold increase in volume fractions and size indices of Golgi complexes and secondary lysosomes was observed, except that monensin had no significant effect on these parameters of secondary lysosomes. These results suggest that the inhibition of VLDL secretion by chloroquine, verapamil and monensin which takes place in the Golgi complex might be due to disruption of trans-membrane proton gradients. An increase in pH of acidic Golgi vesicles may cause swelling and disturb sorting and membrane flow through this organelle.