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Biomedical subjects

J K Horton

Publications and source records attributed to J K Horton.

At least 19 recordsLinked to original sources
PubMed2001

Photoaffinity labeling of mouse fibroblast enzymes by a base excision repair intermediate. Evidence for the role of poly(ADP-ribose) polymerase-1 in DNA repair.

To examine the interaction of mammalian base excision repair (BER) enzymes with DNA intermediates formed during BER, we used a novel photoaffinity labeling probe and mouse embryonic fibroblast cellular extracts. The probe was formed in situ, using an end-labeled oligonucleotide containing a synthetic abasic site; this site was incised by apurinic/apyrimidinic endonuclease creating a nick with 3'-hydroxyl and 5'-reduced sugar phosphate groups at the margins, and then a dNMP carrying a photoreactive adduct was added to the 3'-hydroxyl group. With near-UV light (312 nm) exposure of the extract/probe mixture, six proteins were strongly labeled. Four of these include poly(ADP-ribose) polymerase-1 (PARP-1) and the BER participants flap endonuclease-1, DNA polymerase beta, and apurinic/apyrimidinic endonuclease. The amount of the probe cross-linked to PARP-1 was greater than that cross-linked to the other proteins. The specificity of PARP-1 labeling was examined using various competitor oligonucleotides and DNA probes with alternate structures. PARP-1 labeling was stronger with a DNA representing a BER intermediate than with a nick in double-stranded DNA. These results indicate that proteins interacting preferentially with a photoreactive BER intermediate can be selected from the crude cellular extract.

Animals↗
PubMed2000

The lyase activity of the DNA repair protein beta-polymerase protects from DNA-damage-induced cytotoxicity.

Small DNA lesions such as oxidized or alkylated bases are repaired by the base excision repair (BER) pathway. BER includes removal of the damaged base by a lesion-specific DNA glycosylase, strand scission by apurinic/apyrimidinic endonuclease, DNA resynthesis and ligation. BER may be further subdivided into DNA beta-polymerase (beta-pol)-dependent single-nucleotide repair and beta-pol-dependent or -independent long patch repair subpathways. Two important enzymatic steps in mammalian single-nucleotide BER are contributed by beta-pol: DNA resynthesis of the repair patch and lyase removal of 5'-deoxyribose phosphate (dRP). Fibroblasts from beta-pol null mice are hypersensitive to mono-functional DNA-methylating agents, resulting in increases in chromosomal damage, apoptosis and necrotic cell death. Here we show that only the dRP lyase activity of beta-pol is required to reverse methylating agent hypersensitivity in beta-pol null cells. These results indicate that removal of the dRP group is a pivotal step in BER in vivo. Persistence of the dRP moiety in DNA results in the hypersensitivity phenotype of beta-pol null cells and may signal downstream events such as apoptosis and necrotic cell death.

Cell Line↗
PubMed2000

Protection against methylation-induced cytotoxicity by DNA polymerase beta-dependent long patch base excision repair.

Using a plasmid-based uracil-containing DNA substrate, we found that the long patch base excision repair (BER) activity of a wild-type mouse fibroblast extract was partially inhibited by an antibody to DNA polymerase beta (beta-pol). This suggests that beta-pol participates in long patch BER, in addition to single-nucleotide BER. In single-nucleotide BER, the deoxyribose phosphate (dRP) in the abasic site is removed by the lyase activity of beta-pol. Methoxyamine (MX) can react with the aldehyde of an abasic site, making it refractory to the beta-elimination step of the dRP lyase mechanism, thus blocking single-nucleotide BER. MX exposure sensitizes wild-type, but not beta-pol null mouse embryonic fibroblasts, to the cytotoxic effects of methyl methanesulfonate (MMS) and methylnitrosourea. Expression of beta-pol in the null cells restores the ability of MX to modulate sensitivity to MMS. The beta-pol null cells are known to be hypersensitive to MMS and methylnitrosourea, and in the presence of MX (i.e. under conditions where single-nucleotide BER is blocked) the null cells are still considerably more sensitive than wild-type. The data are consistent with a role of beta-pol in long patch BER, which helps protect cells against methylation damage-induced cytotoxicity.

Alkylating Agents↗
PubMed2000

Acquired alkylating drug resistance of a human ovarian carcinoma cell line is unaffected by altered levels of pro- and anti-apoptotic proteins.

In a systematic study to elucidate the involvement of pro- and anti-apoptotic proteins in alkylating drug resistance of tumor cells, we utilized the A2780(100) line, that was selected by repeated exposure of A2780 cell line (human ovarian carcinoma line) to chlorambucil (CBL). A2780(100) was 5 - 10-fold more resistant to nitrogen mustards (IC50 of 50 - 60 microM) and other DNA crosslinking agents, e.g., cisplatin, and also to DNA topoisomerase inhibitor etoposide (ETO) than A2780. CBL (125 microM) induced extensive apoptosis in A2780 associated with mitochondrial damage but not in A2780(100). No significant differences were observed between A2780 and A2780(100) cells in the basal levels, or the enhanced levels in some cases after CBL treatment, of DNA repair proteins involved in repair of alkyl base adducts or in repair of DNA crosslinks or double strand break repair. However, the basal levels of anti-apoptotic proteins Bcl-xL and Mcl-1 were 4 - 8-fold higher in A2780(100) than in A2780 neither of which expressed Bcl-2. In contrast, the levels of pro-apoptotic Bax and Bak were 3 - 5-fold higher in the CBL-treated A2780 but not in A2780(100). ETO (5 microM) induced apoptosis in A2780 without altering the levels of Bax and Bak in these cells. At the same time, neither overexpression of Bcl-xL in A2780, nor its antisense expression in A2780(100), and nor overexpression of Bax in A2780(100), significantly affected drug sensitivity of either line. Our results suggest that a change in an early step in DNA damage processing which affects intracellular signaling, such as enhanced DNA double-strand break repair, could be the primary cause for development of resistance in A2780(100) cells to drugs which induce DNA crosslinks or double strand-breaks.

Antineoplastic Agents, Alkylating↗
PubMed1999

Mechanism of inhibition of P-glycoprotein-mediated drug transport by protein kinase C blockers.

P-glycoprotein is a membrane ATPase that transports drugs out of cells and confers resistance to a variety of chemically unrelated drugs (multidrug resistance). P-glycoprotein is phosphorylated by protein kinase C (PKC), and PKC blockers reduce P-glycoprotein phosphorylation and increase drug accumulation. These observations suggest that phosphorylation of P-glycoprotein stimulates drug transport. However, there is evidence that PKC inhibitors directly interact with P-glycoprotein, and therefore the mechanism of their effects on P-glycoprotein-mediated drug transport and the possible role of phosphorylation in the regulation of P-glycoprotein function remain unclear. In the present work, we studied the effects of different kinds of PKC inhibitors on drug transport in cells expressing wild-type human P-glycoprotein and a PKC phosphorylation-defective mutant. We demonstrated that PKC blockers inhibit drug transport hy mechanisms independent of P-glycoprotein phosphorylation. Inhibition by the blockers occurs by (i) direct competition with transported drugs for binding to P-glycoprotein, and (ii) indirect inhibition through a pathway that involves PKC inhibition, but is independent of P-glycoprotein phosphorylation. The effects of the blockers on P-glycoprotein phosphorylation do not seem to play an important role, but the PKC-signaling pathway regulates P-glycoprotein-mediated drug transport.

3T3 Cells↗
PubMed1999

Characterization of a chlorambucil-resistant human ovarian carcinoma cell line overexpressing glutathione S-transferase mu.

Ovarian carcinoma cells 10-fold resistant to the alkylating agent chlorambucil (CBL) were isolated after repeated exposure of the parent cells to gradually escalating concentrations of the drug. The resistant variant, A2780(100), was highly cross-resistant (9-fold) to melphalan and showed lower-level resistance to other cross-linking agents. The resistant A2780(100) cells had almost 5-fold higher glutathione S-transferase (GST) activity than the parental A2780 cells with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The pi-class GST(s) was the major isoform(s) in both cell lines. However, the resistant A2780(100) cells had at least 11-fold higher GST mu as compared with the parental cells, in which this isoform was barely detectable. A significant induction of GST mu was observed in A2780 cells, but not in the resistant cells, 18 hr after a single exposure to 100 microM CBL. The induction of GST mu by CBL was both time- and concentration-dependent. Assays of the conjugation of CBL with GSH showed that the human mu-class GST had 3.6- and 5.2-fold higher catalytic efficiency relative to the pi- and alpha-class GSTs, respectively. This difference was reflected in the relatively higher (about 6-fold) efficiency of CBL conjugation in A2780(100) cells as compared with the parental cells. These results have demonstrated for the first time a near-linear correlation between CBL resistance and overexpression of mu-class GSTs and suggest that this overexpression maybe responsible, at least in part, for the acquired resistance of ovarian carcinoma cells to CBL, and possibly the other bifunctional alkylating agents. Consistent with this hypothesis, we found evidence for decreased formation of DNA lesions in A2780(100) compared with the drug-sensitive A2780 cells after exposure to CBL.

Antineoplastic Agents, Alkylating↗
PubMed1999

Intracellular measurement of prostaglandin E2: effect of anti-inflammatory drugs on cyclooxygenase activity and prostanoid expression.

Cyclooxygenase (COX) converts arachidonic acid to prostaglandin (PG) H2, which is further metabolized to various prostaglandins, prostacyclin and thromboxane A2. COX exists in at least two different isoforms. COX-1 is constitutively expressed, whereas COX-2 is induced by proinflammatory stimuli. Prostaglandin E2 is a major metabolite of COX activation. In order to compare the activity of target ligands and COX inhibitors on PGE2 synthesis and release, the responsiveness of several cell lines to the calcium ionophore A23187, bacterial lipopolysaccharide (LPS), nonsteroidal anti-inflammatory drugs (NSAIDs), and the glucocorticoid, dexamethasone, were investigated. For intracellular measurements, the culture supernatant was aspirated, and the cells were thoroughly washed and lysed with dodecyltrimethylammonium bromide. Intracellular and secreted PGE2 were measured with an enzyme immunoassay. A23187 and LPS increased intracellular PGE2 in a dose-dependent manner. Kinetic experiments with A23187-stimulated mouse 3T3 fibroblast cells revealed a distinct biphasic response in COX activity. In the presence of NSAIDs or dexamethasone, there was a dose-dependent inhibition in intracellular PGE2 with A23187-stimulated 3T3 cells. Inhibitory studies demonstrated an apparent increased sensitivity of COX activity to the action of inhibitors when measuring intracellular PGE2 compared with using cell culture supernatants. Indeed, intracellular PGE2 levels were comprehensively reduced in the presence of low concentrations of inhibitor. The utilization of cell culture lysates and, in particular, measurement of intracellular PGE2 should prove useful for identifying new COX inhibitors.

3T3 Cells↗
PubMed1998

Up-regulation of base excision repair correlates with enhanced protection against a DNA damaging agent in mouse cell lines.

DNA polymerase beta is required in mammalian cells for the predominant pathway of base excision repair involving single nucleotide gap filling DNA synthesis. Here we examine the relationship between oxidative stress, cellular levels of DNA polymerase beta and base excision repair capacity in vitro , using mouse monocytes and either wild-type mouse fibroblasts or those deleted of the DNA polymerase beta gene. Treatment with an oxidative stress-inducing agent such as hydrogen peroxide, 3-morpholinosydnonimine, xanthine/xanthine oxidase or lipopolysaccharide was found to increase the level of DNA polymerase beta in both monocytes and fibroblasts. Base excision repair capacity in vitro , as measured in crude cell extracts, was also increased by lipopolysaccharide treatment in both cell types. In monocytes lipopolysaccharide-mediated up-regulation of the base excision repair system correlated with increased resistance to the monofunctional DNA alkylating agent methyl methanesulfonate. By making use of a quantitative PCR assay to detect lesions in genomic DNA we show that lipopolysaccharide treatment of fibroblast cells reduces the incidence of spontaneous DNA lesions. This effect may be due to the enhanced DNA polymerase beta-dependent base excision repair capacity of the cells, because a similar decrease in DNA lesions was not observed in cells deficient in base excision repair by virtue of DNA polymerase beta gene deletion. Similarly, fibroblasts treated with lipopolysaccharide were more resistant to methyl methanesulfonate than untreated cells. This effect was not observed in cells deleted of the DNA polymerase beta gene. These results suggest that the DNA polymerase beta-dependent base excision repair pathway can be up-regulated by oxidative stress-inducing agents in mouse cell lines.

Animals↗
PubMed1998

CXC chemokines suppress proliferation of myeloid progenitor cells by activation of the CXC chemokine receptor 2.

IL-8 is one of the major mediators of the transendothelial migration of neutrophils from the circulation to the site of injury and infection. In this work we demonstrate that the CXC or alpha-chemokines, IL-8 and melanoma growth stimulatory activity (MGSA) induce myeloid suppression via direct action on progenitor cells, mediated by activation of the murine homologue of the CXC chemokine receptor-2 (CXCR2) or IL-8R B. We first show that proliferation of the IL-3-dependent murine myeloid progenitor cell line 32D is suppressed by human IL-8 and the functionally and structurally related peptide, MGSA. Second, we show for the first time the high endogenous expression of the murine CXCR2 in 32D cells, as demonstrated by Northern blot analysis, binding to [125I]macrophage inflammatory protein-2, and macrophage inflammatory protein-2-induced calcium responses in 32D cells. Third, we demonstrate that IL-8 and MGSA induce a rise in intracellular calcium in 32D cells. The IL-8-induced Ca2+ response is desensitizing, since a second dose of IL-8 did not trigger a second calcium response. Other chemokines, including neutrophil-activating protein-2, platelet factor-4, RANTES, and macrophage chemotactic protein-1, neither suppressed the proliferation of 32D cells nor induced a rise in intracellular calcium. Finally, the IC50 of IL-8- and MGSA-dependent suppression of proliferation of 32D cells is in good agreement with the EC50 of IL-8- and MGSA-dependent activation of neutrophil Mac-1 up-regulation and chemotaxis. Our studies are consistent with the idea that IL-8 and MGSA suppress the proliferation of 32D cells by activation of murine CXCR2.

Animals↗
PubMed1998

Swelling-activated chloride currents in a drug-sensitive cell line and a P glycoprotein-expressing derivative are underlied by channels with the same pharmacological properties.

It has been proposed that P glycoprotein (Pgp) expression is associated with swelling-activated Cl- currents in multidrug-resistant cells. The Pgp substrate vinblastine and the modulator verapamil produced a reversible concentration-dependent block of swelling-activated Cl- currents in both a drug-sensitive cell line (MCF-7) and a Pgp-expressing derivative (BC19/3). The similarity of the results obtained in both cell lines suggests that the mechanism of block is not related to Pgp expression and supports the hypothesis that Pgp expression is not necessary for the swelling activation of Cl- currents. In contrast to the results obtained with vinblastine, two other cytoskeleton-disrupting agents, colchicine and cytochalasin D, were not able to affect the swelling-activated Cl- currents in either cell line. The data provided no evidence for the involvement of the cytoskeleton in the swelling activation of Cl- channels in these cell lines. The Cl- channel blockers, 5-nitro-2-(3-phenylpropylamino)benzoic acid and 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, each produced a similar reversible concentration-dependent block in the swelling-activated currents in both the Pgp-expressing and nonexpressing cells. This strongly suggests that the Cl- channel(s) responsible for the swelling-dependent current in both cell lines are the same and, since MCF-7 cells do not express Pgp, that Pgp is not the channel responsible for the volume-activated Cl- currents in these cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Characterization of 2-chloro-N10-substituted phenoxazines for reversing multidrug resistance in cancer cells.

Twenty-one 2-chloro-N10-substituted phenoxazines have been synthesized and characterized as potential modulators of multidrug resistance (MDR). Many of the compounds, at a concentration of 100 microM, enhanced accumulation of vinblastine (VLB) in drug-resistant KB8-5 cells to a greater extent than the same concentration of verapamil (VRP). However, the effects on VLB accumulation were specific, because these derivatives had little activity in the parental drug-sensitive line KB3-1. The compounds slowed the efflux of VLB from KB8-5 cells, suggesting that the chlorophenoxazines, like VRP, can inhibit P-glycoprotein (P-gp)-mediated efflux of VLB from this cell line. Two of the chlorophenoxazine derivatives, and also VRP, were able to stimulate the vanadate-sensitive ATPase activity attributable to P-gp in membranes isolated from MDR1 baculovirus-infected Sf9 cells. This result suggests that these modulators exert their effect by directly interacting with P-gp. Apart from the parent unsubstituted molecule, 2-chlorophenoxazine, there was a good correlation between log10P and the ability of the compounds to enhance VLB accumulation in KB8-5. This suggests that lipophilicity of a modulator is important, but is not the sole determinant of potency. Within this series of compounds, the optimal structural features for MDR modulation include a hydrophobic phenoxazine ring with a -Cl atom in the C-2 position and a tertiary amine group four carbons from the tricyclic ring. Many of the agents at the IC10 concentration completely reversed the 37-fold VLB resistance in KB8-5 cells. The most active agents in KB8-5 were able to partially reverse VLB resistance in an MDR colon carcinoma cell line GC3/c1 and completely reversed the 86-fold VLB resistance in the MDR1-overexpressing breast carcinoma cell line BC19/3. These same agents could only partially sensitize BC19/3 cells to taxol and doxorubicin, suggesting that the chlorophenoxazine derivatives show some specificity for modulating VLB resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Characterization of a novel bisacridone and comparison with PSC 833 as a potent and poorly reversible modulator of P-glycoprotein.

Novel compounds, composed of two acridone moieties connected by a propyl or butyl spacer, were synthesized and tested as potential modulators of P-glycoprotein (P-gp)-mediated multidrug resistance. The propyl derivative 1,3-bis(9-oxoacridin-10-yl)-propane (PBA) was extremely potent and, at a concentration of 1 microM, increased steady state accumulation of vinblastine (VLB) approximately 9-fold in the multidrug-resistant cell line KB8-5. In contrast to the readily reversible effects of VRP and cyclosporin A on VLB uptake and similar to the effects of the cyclosporin analog PSC 833, this modulation by PBA was not fully reversed 6-8 hr after transfer of cells to PBA-free medium. Continuous exposure to 3 microM PBA was nontoxic and could completely reverse VLB resistance in KB8-5 cells. Consistent with its effects on VLB transport, the drug resistance-modulating effect of PSC 833 was significantly more persistent than that of VRP. However, the effect of PBA was, like that of VRP, rapidly reversed once the modulator was removed from the extracellular environment. PBA was able to compete with radiolabeled azidopine for binding to P-gp and to stimulate P-gp ATPase activity. However, both the steady state accumulation of PBA and the rate of efflux of PBA were similar in drug-sensitive KB3-1 and drug-resistant KB8-5 cells, suggesting that this compound is not efficiently transported by P-gp. These results indicate that PBA represents a new class of potent and poorly reversible synthetic modulators of P-gp-mediated VLB transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Requirement of mammalian DNA polymerase-beta in base-excision repair.

Synthesis of DNA by DNA polymerase-beta is distributive on single-stranded DNA templates, but short DNA gaps with a 5' PO4 in the gap are filled processively to completion. In vitro studies have suggested a role of beta-polymerase in different types of DNA repair. However, the significance of these studies to the in vivo role of beta-polymerase has remained unclear. Because genetic studies are essential for determining the physiological role of a gene, we established embryonic fibroblast cell lines homozygous for a deletion mutation in the gene encoding DNA polymerase-beta. Extracts from these cell lines were found to be defective in uracil-initiated base-excision repair. The beta-polymerase-deleted cells are normal in viability and growth characteristics, although they exhibit increased sensitivity to monofunctional DNA-alkylating agents, but not to other DNA-damaging agents. Both the deficiency in base-excision repair and hypersensitivity to DNA-alkylating agents are rescued following stable transfection with a wild-type beta-polymerase minitransgene. These studies demonstrate that beta-polymerase functions specifically in base-excision repair in vivo.

Animals↗
PubMed1995

Strategic down-regulation of DNA polymerase beta by antisense RNA sensitizes mammalian cells to specific DNA damaging agents.

Previously, mouse NIH 3T3 cells were stably transfected with human DNA polymerase beta (beta-pol) cDNA in the antisense orientation and under the control of a metallothionein promoter [Zmudzka, B.Z. and Wilson, S.H. (1990) Som. Cell Mol. Gen., 16, 311-320]. To assess the feasibility of enhancing the efficacy of chemotherapy by an antisense approach and to confirm a role for beta-pol in cellular DNA repair, we looked for increased sensitivity to DNA damaging agents under conditions where beta-pol is down-regulated in the antisense cell line. Such a sensitization is anticipated only where beta-pol is rate-limiting in a DNA repair pathway. A number of agents were tested: cis-diamminedichloroplatinum II (cisplatin); 1,3-bis(2-chloroethyl)-1- nitrosourea (BCNU); ionizing radiation and the radio-mimetic drug bleomycin; the bifunctional alkylating agents nitrogen mustard and L-phenylalanine mustard (melphalan); the monofunctional alkylating agent methyl methane sulfonate (MMS) and ultraviolet (UV) radiation. In the cases of cisplatin and UV radiation, a significant enhancement of cytotoxicity was observed. Damage as a result of both of these agents is thought to be repaired by the nucleotide excision repair (NER) pathway. The results suggest that, in this cell line, beta-pol is involved in and is rate-limiting in NER. We propose that down-regulation of beta-pol by antisense approaches might be used to enhance the cytotoxic effects of cisplatin and other DNA damaging chemotherapeutic agents.

3T3 Cells↗
PubMed1995

Cross-resistance to camptothecin analogues in a mitoxantrone-resistant human breast carcinoma cell line is not due to DNA topoisomerase I alterations.

We have previously described a mitoxantrone-resistant human breast carcinoma cell line, MCF7/MX, in which resistance was associated with a defect in the energy-dependent accumulation of mitoxantrone in the absence of P-glycoprotein overexpression (M. Nakagawa et al., Cancer Res. 52: 6175-6181, 1992). We now report that this cell line is highly cross-resistant to the camptothecin analogues topotecan (180-fold), 9-aminocamptothecin (120-fold), CPT-11 (56-fold), and SN38 (101-fold), but is only mildly cross-resistant to the parent compound camptothecin (3.2-fold) and 10,11-methylenedioxy-camptothecin (2.9-fold). Topotecan accumulation was decreased in MCF7/MX cells compared to parental MCF7/WT cells, and there was a corresponding reduction in topotecan-mediated stimulation of the enzyme/DNA complex formation in MCF7/MX cells compared to MCF7/WT cells. No overexpression of the multidrug resistance-associated protein was detected compared to parental MCF7/WT cells. Furthermore, both sensitive MCF7/WT and mitoxantrone-resistant MCF7/MX cells contain equal amounts of DNA topoisomerase I protein, and DNA relaxation activities were equal in both cell lines and inhibited to the same extent by topotecan and camptothecin. Thus, these results suggest a novel mechanism of resistance to topoisomerase I inhibitors in cancer cells.

Antineoplastic Agents↗
PubMed1994

Unidirectional fluxes of rhodamine 123 in multidrug-resistant cells: evidence against direct drug extrusion from the plasma membrane.

P-glycoprotein (Pgp), a plasma membrane protein overexpressed in multidrug-resistant tumor cells, is an ATPase thought to actively export cytotoxic drugs. It has been proposed that Pgp transports drugs directly from the lipid bilayer to the external medium ("vacuum cleaner" hypothesis). A possible mechanism for this model is that the Pgp is a flippase--i.e., it catalyzes the translocation of hydrophobic substrates from the inner to the outer leaflet of the cell membrane. Two immediate predictions of the vacuum cleaner and flippase hypotheses are that the apparent unidirectional influx of substrate should be less in Pgp-expressing than in Pgp-lacking cells and that this difference should be abolished by inhibition of the Pgp. We used Chinese hamster fibroblasts with different levels of Pgp expression to measure true unidirectional fluxes of rhodamine 123 (R123), a Pgp-transported fluorescent dye that accumulates in mitochondria (hence, its cytosolic concentration remains low at short times after external addition). The unidirectional efflux of R123 was proportional to the level of Pgp expression and was reduced by Pgp inhibitors. The unidirectional influx of R123 was the same in sensitive and resistant cells--i.e., independent of the level of Pgp expression and insensitive to inhibitors of R123 efflux. From these results, we rule out the vacuum cleaner and flippase hypotheses and conclude that Pgp extracts the actively transported substrates from the cytosol and not from the plasma membrane.

ATP Binding Cassette Transporter, Subfamily B, Mem↗