Overexpression of GRP78/BiP in P-Glycoprotein-Positive L1210 Cells is Responsible for Altered Response of Cells to Tunicamycin as a Stressor of the Endoplasmic Reticulum.

P-glycoprotein (P-gp, ABCB1 member of the ABC (ATP-binding cassette) transporter family) localized in leukemia cell plasma membranes is known to reduce cell sensitivity to a large but well-defined group of chemicals known as P-gp substrates. However, we found previously that P-gp-positive sublines of L1210 murine leukemia cells (R and T) but not parental P-gp-negative parental cells (S) are resistant to the endoplasmic reticulum (ER) stressor tunicamycin (an N-glycosylation inhibitor). Here, we elucidated the mechanism of tunicamycin resistance in P-gp-positive cells. We found that tunicamycin at a sublethal concentration of 0.1 µM induced retention of the cells in the G1 phase of the cell cycle only in the P-gp negative variant of L1210 cells. P-gp-positive L1210 cell variants had higher expression of the ER stress chaperone GRP78/BiP compared to that of P-gp-negative cells, in which tunicamycin induced larger upregulation of CHOP (C/EBP homologous protein). Transfection of the sensitive P-gp-negative cells with plasmids containing GRP78/BiP antagonized tunicamycin-induced CHOP expression and reduced tunicamycin-induced arrest of cells in the G1 phase of the cell cycle. Taken together, these data suggest that the resistance of P-gp-positive cells to tunicamycin is due to increased levels of GRP78/BiP, which is overexpressed in both resistant variants of L1210 cells.


Introduction
The endoplasmic reticulum (ER) is a multifunctional membrane organelle with a complex interconnected structure that is involved in intracellular signal transduction [1] and protein synthesis, folding, modification, and quality control [2]. The processes of protein folding and maturation in the ER are controlled by accurate and strictly regulated mechanisms that depend on differential ER homeostasis, such as calcium storage/release equilibrium or equilibrium in pro-/antioxidant status. This homeostasis depends on the energy abundance/deprivation state of the cells and reflects metabolic stimulation, alterations in glycosylation, activation of inflammatory processes, and increases in protein synthesis. Misbalance in this complicated system leads to an increase in misfolded proteins in the cell [3], which is responsible for the development of ER stress in response to the accumulation of

Western Blotting
The protein levels were semiquantitatively determined by Western blotting. Cells were harvested and lysed with SoluLyse reagent containing a protease inhibitor cocktail (both from Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 12,000× g for 10 min. Protein lysates (30 µg per lane) were separated by SDS-PAGE on a Mini-Protean gel electrophoresis system (Bio-Rad, Philadelphia, PA, USA). Proteins were transferred by electroblotting to a polyvinylidene fluoride membrane (GE Healthcare Europe GmbH, Vienna, Austria) and identified by using the following primary and secondary antibodies: rabbit polyclonal primary antibodies against GRP78/BiP, GRP94, IRE1α, ATF6, PERK, CHOP, Bcl-2, Bax, cyclin D1, CNX, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), all from Santa Cruz Biotechnology (Dallas, TX, USA); monoclonal primary antibodies against ATF4 and caspases 3 and 9 from Cell Signaling Technology, Inc. (Beverly, MA, USA); and goat antimouse/rabbit secondary antibody linked with horseradish peroxidase from Santa Cruz Biotechnology. The proteins were visualized with an enhanced chemiluminescence detection system (GE Healthcare Europe GmbH, Vienna, Austria) using an Amersham Imager 600 (GE Healthcare). Broad range protein molecular weight markers (Thermo Fisher Scientific, Bremen, Germany) were used for molecular weight estimations. The intensity of protein bands was quantified by densitometry by using Image Amersham™ image analysis software (GE Healthcare Europe GmbH, Vienna, Austria). All samples were analyzed in triplicate, and the intensity levels were normalized to GAPDH as a housekeeping protein. Significance was established using an unpaired Student's t-test. Quantities of CHOP were also determined in S cells (untransfected and transfected with either control plasmid or plasmid encoding GRP78/BiP), which were processed by same protocol as S, R, and T cell.

Determination of ER Stress-Induced Factors and Chaperones by RT-PCR
Total mRNA was isolated from S, R, and T cells using TRI reagent ® (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. DNA removal was performed with DNase I (Thermo Fisher Scientific, Bremen, Germany), and reverse transcription of total RNA was performed by using a RevertAid™ H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Bremen, Germany) according to the manufacturer's protocol. PCR was performed in a total volume of 25 µL using DreamTaq Green PCR Master Mix according to the manufacturer's protocol (Thermo Fisher Scientific, Bremen, Germany). GAPDH expression was used as an internal standard. After heating at 94 • C for 3 min to inactivate the reverse transcriptase, the samples were subjected to 30 cycles of denaturation (95 • C, 45 s), annealing (58 • C for ATF6, PERK, IRE1α, CHOP, XBP1, GRP94, Bcl-2, Bax, cyclin D1, cyclin B, CNX and cyclin A; 57 • C for cyclin E; and 56.6 • C for GAPDH; 30 s) and extension (72 • C, 90 s), followed by a final extension at 72 • C for 10 min.

Determination of Stress Response Proteins by qPCR
Total mRNA was isolated from L1210 cells using TRI reagent (MRC, USA) according to the manufacturer's instructions. Reverse transcription was performed using the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Bremen, Germany) according to the manufacturer's protocol. Primers and cDNA samples were mixed with iTaq Universal SYBR Green Supermix (Bio-Rad, Laboratories, USA) for qPCR. For the thermal cycle reactions, a CFX96 Real-Time System C1000 Touch Thermal Cycler (BioRad, Laboratories, USA) was used at 95 • C for 10 min and then 39 cycles at 95 • C for 10 s and at 55 • C for 30 s. The relative amount for each transcript was calculated by a standard curve of cycle thresholds for cDNA samples and normalized to the amount Cells 2020, 9, 890 6 of 23 of β-actin. The polymerase chain reaction (PCR) was performed in triplicate for each sample, after which all experiments were repeated twice. The data were analyzed with Bio-Rad CFX96T software. Baseline levels for each gene were computed automatically. The results were quantified from Ct values according to the formula ∆∆Ct = ∆Ct sample-∆c housekeeping gene. Quantities of GRP78/BiP transcripts were also determined in S cells (untransfected and transfected with either control plasmid or plasmid encoding GRP78/BiP), which were processed by same protocol as S, R, and T cell.

Visualization of GRP78/BiP in S, R, and T Cells Using Immunofluorescence Confocal Microscopy
After culture, the cells were washed and resuspended in PBS and then transferred onto poly-L-lysine glass slides (Menzel-Glaser, Braunschweig, Germany). The bound cells were washed twice in PBS, fixed with cooled methanol (−20 • C) for 20 min, washed in PBS, and then blocked with 0.2% bovine serum albumin (BSA) in PBS for 1 h at 37 • C. The cells were then incubated with primary antibody against GRP78/BiP diluted 1:1000 for 1 h at 37 • C in PBS containing 0.2% BSA, washed twice in PBS containing 0.2% BSA, and then incubated with Alexa Fluor 488 donkey anti-goat antibody (Life Technologies Corporation, Eugene, OR, USA) in PBS containing 0.2% BSA for 1 h at 37 • C. The specimens were washed twice in PBS containing 0.2% BSA and were labeled with 10 mg/L 4'-6-diamidino-2-phenylindole (DAPI, Sigma Aldrich, St. Louis, MO, USA) in PBS to visualize the nuclei. Finally, the coverslips were mounted onto slides with a mounting medium (80% glycerol) and analyzed using a confocal laser scanning microscope (Nikon Eclipse Ti, Tokyo, Japan).

Detection of GRP78/Bip on Cell Surface of L1210 cells by Flow Cytometry
L1210 cells were cultivated in the absence or presence of tunicamycin (0.1 µM) for 24 h. Subsequently, the cells were washed three times with PBS, resuspended (5 × 10 5 cells per mL) in RPMI medium with 5% of defatted BSA (Sigma Aldrich, St. Louis, MO, USA), and incubated for 30 min with anti-GRP78 antibody (described in previous chapter) in a humidified atmosphere supplemented with 5% CO 2 at 37 • C. After incubation, the cells were washed three times with RPMI medium containing 5% BSA and subsequently left to interact with secondary antibody (Goat anti-Rabbit IgG linked with Alexa Fluor 660, A21074, Thermo Fisher Scientific, Bremen, Germany). The labeled cells were analyzed on BD Accuri C6 flow cytometer.

Effect of Tunicamycin on S, R, and T Cell Cycle Progression
P-gp-positive R and T cells could be repeatedly cultured in medium containing tunicamycin at concentration 0.1 µM. In contrast, in S cells after the third passage, massive cell death was detected ( Figure S1). However, a single passage in medium containing this concentration of tunicamycin did not induce massive cell death either in P-gp-negative S or P-gp-positive R and T cells ( Figure S1 in Supplementary Materials); therefore, these condition was used in further experiments.
First, we focused on whether a single passage of S, R, and T cells at sublethal concentration of tunicamycin, which did not induce cell death, initiated apoptosis as measured by the FAV/PI kit. Incubation of S, R, and T cells in the presence of 0.1 µM tunicamycin for 4, 8, and 24 h did not induce any increase in the proportion of cells either in apoptosis (stained by FAV) or necrosis (stained with PI), and there were no cells in late apoptosis/necrosis that were stained for both of these markers ( Figure 1A). In the absence of tunicamycin, S cells expressed lower levels of the antiapoptotic Bcl-2 protein and almost identical levels of the proapoptotic Bax protein compared to those of R and T cells at both the mRNA and protein level (Figure 2A,B). A single passage of S, R, and T cells in the presence of tunicamycin did not considerably influence the expression of Bcl-2 and Bax at the mRNA or protein level (Figure 2A  In the absence of tunicamycin, S cells expressed lower levels of the antiapoptotic Bcl-2 protein and almost identical levels of the proapoptotic Bax protein compared to those of R and T cells at both the mRNA and protein level (Figure 2A,B). A single passage of S, R, and T cells in the presence of tunicamycin did not considerably influence the expression of Bcl-2 and Bax at the mRNA or protein level (Figure 2A  Increased levels of the initiating procaspase 9 protein and almost identical levels of the Significance: Data differ from values obtained in cells that were not incubated in the presence of tunicamycin (a) at the levels: * p < 0.02; ** p < 0.002. (C) Activated, proteolytically cleaved caspase 9 (upper) and caspase 3 (lower) as a control for caspase activation in R cells after 10 min of UV irradiation using a germicide lamp: After irradiation, the cells were incubated for 4 and 8 h in culture medium. Similar proteolytically cleaved forms of caspases after UV irradiation were also detected in S and T cells (not shown).
Increased levels of the initiating procaspase 9 protein and almost identical levels of the executioner procaspase 3 protein were detected by Western blotting in S cells compared with those in R and T cells ( Figure 2B). However, culture of S, R, and T cells in the presence of tunicamycin did not induce alterations in the protein levels of either procaspase in S, R, and T cells; moreover, proteolytic cleavage to active caspases was not observed. In the control experiment, we demonstrated this proteolytic activation in S, R, and T cells after exposure to UV irradiation by a germicide lamp (as shown for R cells in Figure 2C). Thus, we may conclude that tunicamycin at a concentration of 0.1 µM does not induce cell death during a 24-h incubation period; therefore, we chose these conditions for subsequent experiments.
Tunicamycin at a concentration of 0.1 µM induced an increase in the proportion of cells in the G1 phase of the cell cycle, which was associated with a decrease in the proportion of cells in the S and G2/M phases in S cells ( Figure 3). However, in both P-gp-positive cells (R and T), retention of cells in the G1 phase was much less pronounced ( Figure 3).    . Expression of cyclin D1 in the presence or absence of tunicamycin: Respective mRNAs were detected by RT-PCR (A) and proteins were detected by Western blotting (B). S, R, and T cells were incubated in the presence or absence of tunicamycin for 4, 8, and 24 h. Signals for GAPDH mRNA and protein were used as internal controls. Data are representative of three independent measurements. Quantification of mRNA or protein signals obtained by densitometry are summarized in column plots. Data were normalized to GAPDH expression and are expressed as the mean ± S.E.M. of three independent measurements. Significance: Data differ from value obtained without incubation in the presence of tunicamycin (0) at the level * p < 0.05, ** p < 0.02; data differ from that correspondingly obtained for S cells at the level # p < 0.01. Expression of other cyclins A, B1, and E at mRNA levels are documented in the Supplementary Materials ( Figure S2).

Endoplasmic Stress Receptors and Activation of the UPR
GRP78/BiP plays a crucial role in the ER stress response by inactivating endoplasmic reticulum receptors (PERK, IRE1, and ATF6) [6]. P-gp-negative cells contained less GRP78/BiP than P-gppositive R and T cells at the mRNA ( Figure 5A) and protein levels ( Figure 5B). This protein was localized intracellularly and predominantly in structures around the nucleus ( Figure 5C). Moreover, this protein was detected in R and T cells than in S cells. Some GRP78/BiP was also localized in the plasma membrane of cells, particularly in the R and T variants ( Figure 5C). To quantify the cell surface GRP78/BiP, we performed flow cytometry counting of viable S, R, and T cells stained with anti- Quantification of mRNA or protein signals obtained by densitometry are summarized in column plots. Data were normalized to GAPDH expression and are expressed as the mean ± S.E.M. of three independent measurements. Significance: Data differ from value obtained without incubation in the presence of tunicamycin (0) at the level * p < 0.05, ** p < 0.02; data differ from that correspondingly obtained for S cells at the level # p < 0.01. Expression of other cyclins A, B1, and E at mRNA levels are documented in the Supplementary Materials ( Figure S2).

Endoplasmic Stress Receptors and Activation of the UPR
GRP78/BiP plays a crucial role in the ER stress response by inactivating endoplasmic reticulum receptors (PERK, IRE1, and ATF6) [6]. P-gp-negative cells contained less GRP78/BiP than P-gp-positive R and T cells at the mRNA ( Figure 5A) and protein levels ( Figure 5B). This protein was localized intracellularly and predominantly in structures around the nucleus ( Figure 5C). Moreover, this protein was detected in R and T cells than in S cells. Some GRP78/BiP was also localized in the plasma membrane of cells, particularly in the R and T variants ( Figure 5C). To quantify the cell surface GRP78/BiP, we performed flow cytometry counting of viable S, R, and T cells stained with anti-GRP78/BiP antibody ( Figure 5D). These data showed slightly increased amounts of GRP78/BiP on the plasma membrane of R and T cells when compared with that of S cells. Incubation of S cells in tunicamycin-containing medium induced a strong increase in GRP78/BiP (Supplementary Materials Figure S3). Protein levels in cells that were incubated in the presence of tunicamycin for 24 h compared to cells without this treatment increased 10-fold. In P-gp (R and T)-positive cells in which higher levels of GRP78/BiP have been registered in the absence of tunicamycin, its increase is less pronounced and is about 2-fold. and T cells, and these levels were unaffected by incubation of the cells in the presence of 0.1 µM tunicamycin ( Figure 6A,B). However, the 50 kDa ATF6 fragment was present in both the drugsensitive S and drug-resistant R and T cells, indicating the activation of the ATF6 receptor in all L1210 cell variants. This active ATF6 fragment was present in higher levels in S cells than in R and T cells ( Figure 6B). Incubation of S cells in medium containing 0.1 µM tunicamycin did not induce any differences in the levels of the 50 kDa fragment. Increases in this fragment in R and T cells were similar to that of S cells only after incubation in the presence of tunicamycin for 24 h.  Similar levels of mRNA encoding ATF6 and inactive 90 kDa ATF6 protein were found in S, R, and T cells, and these levels were unaffected by incubation of the cells in the presence of 0.1 µM tunicamycin ( Figure 6A,B). However, the 50 kDa ATF6 fragment was present in both the drug-sensitive S and drug-resistant R and T cells, indicating the activation of the ATF6 receptor in all L1210 cell variants.
This active ATF6 fragment was present in higher levels in S cells than in R and T cells ( Figure 6B). Incubation of S cells in medium containing 0.1 µM tunicamycin did not induce any differences in the levels of the 50 kDa fragment. Increases in this fragment in R and T cells were similar to that of S cells only after incubation in the presence of tunicamycin for 24 h.
The PERK receptor mRNA was expressed in all variants of L1210 cells at equal amounts ( Figure  6A). The mRNA level of this receptor was not affected by incubation in medium containing tunicamycin. In S, R, and T cells cultured with tunicamycin, the protein level of PERK increased slightly ( Figure 6B). The downstream target gene of this receptor is the apoptotic transcription factor CHOP. The level of CHOP mRNA ( Figure 6A) increased in sensitive cells cultured with 0.1 µM tunicamycin (namely after 4 h). In R and T cells, the increase in CHOP mRNA was detectable only after 24-h culture of cells in tunicamycin. Higher level of CHOP mRNA was detected in S cells than in R and T cells by qPCR (Figure 8). Moreover, strong increases in this transcript due to incubation in medium containing tunicamycin were detectable only in S cells. Increases in CHOP levels induced by tunicamycin were maximally detectable in S cells by Western blotting after 8 h of culture ( Figure  6B). In resistant R and T cells, small but detectable amounts of CHOP protein were measured only after 24 h of culture in medium containing tunicamycin.  The PERK receptor mRNA was expressed in all variants of L1210 cells at equal amounts ( Figure 6A). The mRNA level of this receptor was not affected by incubation in medium containing tunicamycin. In S, R, and T cells cultured with tunicamycin, the protein level of PERK increased slightly ( Figure 6B). The downstream target gene of this receptor is the apoptotic transcription factor CHOP. The level of CHOP mRNA ( Figure 6A) increased in sensitive cells cultured with 0.1 µM tunicamycin (namely after 4 h). In R and T cells, the increase in CHOP mRNA was detectable only after 24-h culture of cells in tunicamycin. Higher level of CHOP mRNA was detected in S cells than in R and T cells by qPCR (Figure 8). Moreover, strong increases in this transcript due to incubation in medium containing tunicamycin were detectable only in S cells. Increases in CHOP levels induced by tunicamycin were maximally detectable in S cells by Western blotting after 8 h of culture ( Figure 6B). In resistant R and T cells, small but detectable amounts of CHOP protein were measured only after 24 h of culture in medium containing tunicamycin.
The expression of IRE1 was present in all three variants of L1210 cells at the mRNA and protein levels ( Figure 6). While in S cells incubated in medium with tunicamycin there was a slight increase in IRE1 expression at the mRNA and protein levels, such effects were not observed in R and T cells. IRE1 seemed to be activated because the product of its unconventional RNA splicing activity, spliced XBP1 mRNA [45], was present in all three cell variants ( Figure 7A). Therefore, we used special qPCR primers for quantification of the unspliced (uXBP1), spliced (sXBP1), and total (tXBP1) variants of XBP1 mRNA (Table 2). While S cells contained higher amounts of uXBP1 and tXBP1 than R and T cells, both P-gp-positive variant L1210 cells (R and T) contained higher amounts of active sXBP1 in the absence of tunicamycin. In sensitive S cells, 0.1 µM tunicamycin induced an increase in the levels of all three XBP1 qPCR products to amounts exceeding the corresponding values observed for R and T cells. An exception to this effect for the sXBP1 variant occurred in S cells after 24 h of incubation in medium containing tunicamycin (Figure 7). The activated sXBP1 variant was maximally detected in S cells at 8 h of culture (Figure 7). The expression of IRE1 was present in all three variants of L1210 cells at the mRNA and protein levels ( Figure 6). While in S cells incubated in medium with tunicamycin there was a slight increase in IRE1 expression at the mRNA and protein levels, such effects were not observed in R and T cells. IRE1 seemed to be activated because the product of its unconventional RNA splicing activity, spliced XBP1 mRNA [45], was present in all three cell variants ( Figure 7A). Therefore, we used special qPCR primers for quantification of the unspliced (uXBP1), spliced (sXBP1), and total (tXBP1) variants of XBP1 mRNA (Table 2). While S cells contained higher amounts of uXBP1 and tXBP1 than R and T cells, both P-gp-positive variant L1210 cells (R and T) contained higher amounts of active sXBP1 in the absence of tunicamycin. In sensitive S cells, 0.1 µM tunicamycin induced an increase in the levels of all three XBP1 qPCR products to amounts exceeding the corresponding values observed for R and T cells. An exception to this effect for the sXBP1 variant occurred in S cells after 24 h of incubation in medium containing tunicamycin (Figure 7). The activated sXBP1 variant was maximally detected in S cells at 8 h of culture (Figure 7).  Table 1: Data are representative of three independent experiments. GAPDH was used as an internal control. (B) qRT-PCR quantification of total, spliced, and unspliced XBP1 transcripts (using primers listed in Table 2) in S, R, and T cells after 0 (a), 4 (b), 8 (c), and 24 (d) hours of incubation in the presence of tunicamycin: Transcript levels were normalized to GAPDH as a housekeeping gene and are expressed as the mean ± S.E.M. of three independent measurements. Transcriptions of all three forms (s, us, and t) obtained for untreated S cells was arbitrarily taken as one. Significance: Data differ from value obtained without incubation in the presence of tunicamycin (a) at the level * p < 0.02, ** p < 0.002.
We also detected lower amounts of the endoplasmic reticulum Ca2+-dependent lectin/chaperone-CNX in R and T cells than in S cells at the protein level ( Figure S5 in Supplementary Materials). However, at the mRNA level, we did not detect differences in CNX expression. GRP94 expression at the mRNA level did not differ in S, R, and T cells, and tunicamycin did not induce  Table 1: Data are representative of three independent experiments. GAPDH was used as an internal control. (B) qRT-PCR quantification of total, spliced, and unspliced XBP1 transcripts (using primers listed in Table 2) in S, R, and T cells after 0 (a), 4 (b), 8 (c), and 24 (d) hours of incubation in the presence of tunicamycin: Transcript levels were normalized to GAPDH as a housekeeping gene and are expressed as the mean ± S.E.M. of three independent measurements. Transcriptions of all three forms (s, us, and t) obtained for untreated S cells was arbitrarily taken as one. Significance: Data differ from value obtained without incubation in the presence of tunicamycin (a) at the level * p < 0.02, ** p < 0.002.  GTC CAT GGG AAG ATG TTC TGG-3  5 -CTG AGT CCG AAT CAG GTG CAG-3   usXBP1 c, us  5 -GTC CAT GGG AAG ATG TTC TGG-3  5 -CAG CAC TCA GAC TAT GTG CA-3  We also detected lower amounts of the endoplasmic reticulum Ca2+-dependent lectin/chaperone-CNX in R and T cells than in S cells at the protein level ( Figure S5 in Supplementary Materials). However, at the mRNA level, we did not detect differences in CNX expression. GRP94 expression at the mRNA level did not differ in S, R, and T cells, and tunicamycin did not induce detectable changes ( Figure S5 in Supplementary Materials). However, at the protein level, GRP94 was upregulated in all L1210 cell variants after 24 h of incubation in medium containing tunicamycin.

Downregulation of CHOP Expression After Transfection of S Cells With GRP78/BiP
The activity of endoplasmic receptors is negatively regulated through interactions with GRP78/BiP [6]. Increases in unfolded proteins within the ER lumen cause dissociation of GRP78/BiP from all three ER membrane receptors, which induces their activation. S cells that contain less GRP78/BiP respond to ER stressors more efficiently than R and T cells, which contain more of this protein ( Figure 5). This may be responsible for arresting S cells in the G1 phase of the cell cycle after incubation with tunicamycin, which is much less pronounced in R and T cells (Figure 3). To explore this idea, we increased the intracellular level of GRP78/BiP in S cells by transfection with the pcDNA3.1(+)-GRP78/BiP plasmid (32701) as previously described [40]. The plasmid pCMV-PL (20783) was used as a negative control. Transfection of S cells with the plasmid encoding GRP78/BiP induced an increase in this protein to levels similar as in R and T cells, and approximately 6-fold increase was achieved compared to non-transfected or transfected control plasmid cells ( Figure 8A). This increase in GRP78/BiP was associated with downregulation of DDIT3 gene (encoding CHOP) transcription in GRP78/BiP-transfected S cells (compared with that of either untransfected cells or cells transfected with the control plasmid) that were cultured in the presence of tunicamycin ( Figure 8B). Nevertheless, levels of this transcript still exceeded levels typical for R and T cells.
Similar to untransfected S cells (Figure 3), S cells transfected with the control plasmid and cultured in the presence of tunicamycin for 24 h induced the retention of cells in the G1 phase of the cell cycle ( Figure 8C). However, in S cells transfected with GRP78/BiP, no similar retention in the G1 phase after culture in tunicamycin was detected ( Figure 8C), and they responded to tunicamycin similarly to R and T cells (Figure 3). Therefore, we conclude that the tunicamycin-induced halt in the cell cycle at the G1 phase observed in S cells was causally linked to a lower level of GRP78/BiP, and the higher levels in R and T cells protected against tunicamycin-induced cell cycle arrest. Consistent with this finding, silencing GRP78/BiP induced cell cycle arrest in the G1 phase [46]. 8B). Nevertheless, levels of this transcript still exceeded levels typical for R and T cells.
Similar to untransfected S cells (Figure 3), S cells transfected with the control plasmid and cultured in the presence of tunicamycin for 24 h induced the retention of cells in the G1 phase of the cell cycle ( Figure 8C). However, in S cells transfected with GRP78/BiP, no similar retention in the G1 phase after culture in tunicamycin was detected ( Figure 8C), and they responded to tunicamycin similarly to R and T cells (Figure 3).  Data are representative of three independent measurements, and GAPDH was used as an internal control. Protein bands were quantified by densitometry, GRP78/BiP was normalized to GAPDH, and the data are expressed as the mean ± S.E.M. of three independent measurements in the column plot. Significance: Data differ from value obtained for untrasfected S cell at the level ** p < 0.002. (B) qRT-PCR quantification of CHOP transcripts in untransfected and transfected S cells, R cells, and T cells (symbols 1-5 are described above). Cells were incubated with tunicamycin for 0 (a), 4 (b), 8 (c), and 24 (d) hours. GAPDH was used as an internal control, and the data are expressed as the mean ± S.E.M. Transcription of DDIT3 gene (encoding CHOP) obtained for untransfected S cells, which were untreated with tunicamycin, was arbitrarily taken as one. Data differ from value obtained without incubation in the presence of tunicamycin (a) at the level # p < 0.02, + p < 0.002; data differ from that correspondingly obtained for S cell transfected with control plasmid

Discussion
Currently, we see an increased interest in the role of ER stress in the development of neoplastic cell resistance to therapeutics. Therefore, there is also an increased interest in the relationships between different MDR mechanisms and ER stress, resulting in their synergy in cellular mechanisms and ensuring reduced cell sensitivity to drugs. We recently published a review paper oriented on this topic [36]. The current paper is motivated by the effort to contribute to the understanding of the complex relationship in this issue. P-gp-positive variant L1210 cells (R and T) strongly expressed P-gp either at the mRNA or protein level compared with that of S cells [38]. Moreover, we used calcein retention assay to evaluate P-gp efflux activities and found that R and T cells possessed P-gp efflux activities but that S cells lacked this activity [33]. Both R and T cells were much less sensitive to P-gp substrates, such as VCR, doxorubicin, and mitoxantrone, than S cells [35]. All these features were periodically controlled in S, R, and T cells in our laboratory.
While R and T cells could be repeatedly cultured in medium containing sublethal concentrations of tunicamycin (0.1 µM), S cells did not endure these conditions well, and after the third passage, massive cell death was detected [33,35]. This result is also documented in the Supplementary Materials ( Figure S1). However, a single passage in medium containing this concentration of tunicamycin did not induce massive cell death either in P-gp-negative S or P-gp-positive R and T cells ( Figure S1 in Supplementary Materials). Consistent with our results, these conditions did not induce massive cell death in various cell models during a single passage, but when tunicamycin was applied at higher concentrations, cell death due to ER stress was detected [47][48][49].
In this study, we aimed to explain why P-gp-positive R and T cells are less sensitive to the ER stressor tunicamycin [33,35,50,51]. After passaging R and T cells with tunicamycin, P-gp protein was detected only in unglycosylated [33,51] and ubiquitinated [50] molecular forms, and this transporter was localized in its typical position in the plasma membrane with sustained calcein-efflux activity.
Another significant change in protein expression is the marked upregulation of Bcl-2 protein in R and T cells compared to S cells (Figure 2). The upregulation of Bcl-2 is often involved in MDR, and the concurrent upregulation of P-gp and Bcl-2 has been previously described in samples obtained from patients with AML and acute lymphoblasticleukemia (ALL) [52,53]. However, neither levels of anti-apoptotic Bcl-2 nor pro-apoptotic Bax protein were altered after culturing S, R, and T cells in the presence of tunicamycin (Figure 2). Reduced levels of procaspase 9 in R and T cells may reflect the antiapoptotic effect of upregulated Bcl-2 [54]. Both of these procaspases must be activated by specific proteolysis in response to the mitochondrial intrinsic apoptotic pathway [55]. However, we did not observe proteolytic activation of both caspases after a single culture of S, R, and T cells in the presence of tunicamycin. Therefore, it can be assumed that, under these conditions, there is no initiation of apoptosis processes, which was demonstrated also by double staining of cells with FAV and PI ( Figure 1).
Although single culture of S cells does not result in apoptosis, these conditions induce cell retention in the G1 phase of the cell cycle. Similar behavior was not observed in R and T cells. The retention in the G1 phase of cell cycle in response to the presence of tunicamycin has been already described, and a time-dependent increase in the cell proportion in this phase in cells of hepatocellular carcinoma (Hep3B) after treatment with tunicamycin can be used as example [56]. However, the question remains why R and T cells are not retained in the G1 phase of the cell cycle after cultivation with tunicamycin. This may be caused (at least partly) by upregulation of cyclin D1 expression (on both mRNA and protein levels; Figure 4) in these P-gp positive cell variants. Cyclin D1 is known to be active in the transition of G1 to S phase of cell cycle [57] Expression of Cyclin E, another cyclin active in this cell cycle transition [58], was also found to be upregulated (at the mRNA level, Supplementary Material Figure S2) in R and T cells as compared to S cells. In contrast, the mRNA levels of cyclin A (which resides in the nucleus during S phase where it is involved in DNA replication [59]) and B1 (a G2/mitotic-specific cyclin [60]) were almost identical in S, R, and T cells, independent of the presence of tunicamycin ( Figure S2 in Supplementary Materials). Therefore, differences in cell cycle regulation between P-gp-positive cells (R and T) and P-gp-negative S cells seem to originate from alterations in the G1/S phase transition. When tunicamycin was not present, the cell cycles of S, R, and T cells behaved similarly, but when tunicamycin was present, R and T cells continued in the cell cycle, in contrast to S cells, which preferentially halted in G1 phase (Figure 3).
By inhibiting the N-glycosylation process of proteins, tunicamycin blocks their folding in the ER and thereby induces accumulation of unfolded proteins. This causes ER stress and finally arrest of cell cycle in G1 phase [21]. GRP78/BiP is a central regulator of the cellular response to stress induced by accumulation of unfolded proteins in a cell [6]. Under these circumstances, GRP78/BiP dissociates from PERK, IRE1, and ATF6, which in turn mediate the response to ER stress. The fact that P-gp positive R and T cells have a higher cellular content of GRP78/BiP (Figure 4) than their P-gp negative counterpart S cells suggests that the former will be less stressed by tunicamycin-induced N-glycosylation retarding and subsequent accumulation of unfolded proteins. A small proportion of GRP78/BiP has been found on the cell surface in addition to its classical intracellular localization (Figure 4). Relocalization of GRP78/BiP from the ER to the plasma membrane was studied in 293T, HeLa, and MCF7 cells, and cell surface GRP78/BiP was assumed to play critical roles in cell signaling, proliferation, and overall survival [61].
Upon release of GRP78/BiP from PERK, IRE1, and ATF6, these receptors activate and mediate their regulatory pathways within the UPR (Figure 9). Activation of PERK, IRE1, and ATF6 in all L1210 cell variants results from the presence of their downstream regulatory molecules: CHOP ( Figure 6) ATF4 ( Figure S4 in Supplementary Materials), spliced XBP1 (Figure 7), and the 50 kDa ATF6 fragment ( Figure 6). The levels of CHOP, ATF4, 50 kDA ATF6, and both the spliced and unspliced forms of the XBP1 transcript are higher in P-gp negative S cells than in P-gp positive R and T cells. This indicates a higher level of ER stress induced by tunicamycin in S cells than in R and T cells. Interestingly, incubation of S cells in the presence of tunicamycin for 24 h leads to a decrease in the unspliced variant of the XBP1 transcript ( Figure 7) and the protein level of CHOP ( Figure 6). This downregulation is not observed for CHOP transcript (Figure 8).
CNX, a protein required for quality control of new proteins in ER, is less present in R and T cells than in S cells ( Figure S5, Supplementary Material). We have previously described similar results comparing S and R cells [62]. Nevertheless, immunoprecipitation showed the existence of a complex of calnexin with P-glycoprotein in R cells, especially after incubation with VCR [63]. Since CNX together with another Ca 2+ -dependent ER lectin/chaperone calreticulin represents a crucial coupled player in protein quality control based on N-glycosylation in the ER [16], it is expected that P-gp-positive R and T cells differ from P-gp-negative cells in this process. Therefore, qualitative and quantitative differences in protein N-glycosylation could occur in L1210 cells when P-gp is overexpressed. Consistent with this, we previously detected differences between P-gp-negative S and P-gp-positive R and T cells in the specific binding of several lectins with cell surface-located sugars [35,38,50,51,64,65]. Culture of S, R, and T cells in the presence of 0.1 µM tunicamycin did not affect the expression of this CNX ( Figure S5 in Supplementary Materials). The last protein that we detected in this set of experiments was GRP94, which is known to act as an antiapoptotic stimulus [15], and its expression on protein level seems to be elevated in S, R, and T cells after cultivation in medium containing tunicamycin ( Figure S5 in Supplementary Material).
In the case of CHOP, as the main inducer of apoptosis associated with ER stress [8], we observed its higher amount in S than in R and T cells. This appears to be responsible for a more pronounced response to tunicamycin in S than in R and T cells. This is consistent with the higher amount of GRP78/BiP in R and T cells than in S cells ( Figure 5), since GRP78/BiP acts against the activation of PERK and the consequent induction of its downstream protein CHOP [8]. However, GRP78/BiP induces the elimination of CHOP by initiating its proteasomal cleavage because GRP78/BiP is known to interact directly with the N-terminal domain of CHOP, which accelerates CHOP ubiquitination and consequent degradation [66]. The latter possibility should eliminate CHOP protein without direct influence on transcription of the CHOP gene. However, the level of CHOP mRNA observed in S, R, and T cells in the absence or presence of tunicamycin seemed to be proportional to the level of CHOP protein ( Figure 6). Thus, we propose that a higher level of GRP78/BiP induces changes in CHOP level between P-gp-positive R and T and P-gp-negative S cells that are likely due to a block in PERK-regulated transcription rather than through the initiation of CHOP proteasomal degradation.
Taken all fact together, we may state that higher levels of GRP78/BiP in P-gp positive than in P-gp negative cells are responsible for sustained deactivation of all three ER receptor (PERK, IRE1, and ATF6), which finally caused the lack of cell cycle arrest in G1 phase in former cells treated by tunicamycin. In accordance with the latest statement are results with S cells transfected with a plasmid encoding GRP78/BiP, in which lower expression of CHOP was achieved, and upon tunicamycin treatment, lack of cell cycle arrest in the G1 phase was observed (Figure 8).

Conclusions
R and T cells with high P-gp expression grow during repeated passages in the presence of 0.1 µM tunicamycin without limitation [33,35,50]. P-gp-negative S cells do not have this ability. Consistently, we observed an increase in the proportion of S cells that were arrested in the G1 phase of the cell cycle after one passage in the presence of tunicamycin, which was not observed in R and T cells (Figure 3).
In the present paper, we have shown that this ability results from changes in the expression of proteins that are involved in the cellular response to tunicamycin-induced accumulation of unfolded proteins during ER stress. The central factor is GRP78/BiP, which is upregulated in R and T cells ( Figure 5) and induced more effective inhibition of the active forms of the three ER stress receptors PERK, ATF-6, and IRE1 ( Figure 9).
tunicamycin. In accordance with the latest statement are results with S cells transfected with a plasmid encoding GRP78/BiP, in which lower expression of CHOP was achieved, and upon tunicamycin treatment, lack of cell cycle arrest in the G1 phase was observed (Figure 8).

Conclusions
R and T cells with high P-gp expression grow during repeated passages in the presence of 0.1 µM tunicamycin without limitation [33,35,50]. P-gp-negative S cells do not have this ability. Consistently, we observed an increase in the proportion of S cells that were arrested in the G1 phase of the cell cycle after one passage in the presence of tunicamycin, which was not observed in R and T cells (Figure 3). In the present paper, we have shown that this ability results from changes in the expression of proteins that are involved in the cellular response to tunicamycin-induced accumulation of unfolded proteins during ER stress. The central factor is GRP78/BiP, which is upregulated in R and T cells ( Figure 5) and induced more effective inhibition of the active forms of the three ER stress receptors PERK, ATF-6, and IRE1 ( Figure 9).

Figure 9.
A schematic describing the altered response of R and T cells to tunicamycin-induced ER stress: In the absence of tunicamycin, all three ER stress receptors (PERK, IRE1, and ATF-6) are blocked by GRP78/BiP and therefore do not undergo downstream processes. In the presence of tunicamycin, when N-glycosylation of newly synthetized proteins is blocked, nonglycosylated/unfolded proteins accumulate in the ER and GRP78/BiP begins to interact with them. In S cells with lower GRP78/BiP levels, the amount of GRP78/BiP is insufficient to simultaneously bind unfolded proteins and to block all three ER receptors. Therefore, GRP78/BiP releases from PERK, IRE1, and ATF-6, and these receptors are activated and induce the following stress response pathways: i. PERK dimerizes, autophosphorylates, and subsequently inactivates eIF2α via specific phosphorylation. This leads to inhibition of eIF2 (alpha, beta, and gamma) heterotrimer formation. This results in a rapid decrease in translation or suppression of global protein synthesis. Due to this action, the arrest of S cells in the G1 phase of the cell cycle occurs. If this stress persists during repeated passages in the presence of tunicamycin, initiation of the PERK-ATF4 pathway induces CHOP expression; ii. IRE1 is homooligomerized, autophosphorylated, and exhibits RNA splicing activity that results in specific splicing of XBP1 mRNA and translation of the spliced variant of this protein. The truncated XBP1 variant is a functional transcription factor that initiates the expression of ER chaperones and endoplasmic reticulum-associated degradation (ERAD) genes. This allows cells to recover from ER stress. During this period, cell arrest occurs in the G1 phase; iii. after GRP78/BiP release, ATF6 is specifically cleaved and a 50-kDa fragment translocates into the nucleus, where it induces transcription of ER chaperones (e.g., GRP78/BiP), protein degradation enzymes, and XBP1. If the effect of sublethal concentrations of tunicamycin does not last long (during the first and second passages), the cells are able to grow normally in subsequent passages in the absence of tunicamycin. However, if this stress-induced condition persists, S cells do not proliferate, and after the third passage in the presence of tunicamycin, only a few viable cells were detected. R and T cells contain more GRP78/BiP, which is able to interact with larger amounts of unfolded proteins without release from the ER stress receptors. Under these circumstances, there is no activation of these receptors upon incubation with tunicamycin, as there is in S cells. Therefore, R and T cells are not retained in the G1 phase of the cell cycle under the influence of tunicamycin. Increasing GRP78/BiP in S cells by transfection with the gene encoding this protein prevented arrest in G1 cell cycle phase in transfected S cells after incubation with tunicamycin.
Tunicamycin induced the accumulation of nonglycosylated (unfolded) proteins to which GRP78/BiP bound upon its release from the ER stress receptors, which were subsequently activated. Cells with higher GRP78/BiP levels tolerate higher levels of unfolded proteins within the ER without activation of ATF-6, PERK, and IRE1 ( Figure 9). Correspondingly, in R and T cells, we observed decreased levels of the downstream regulators CHOP and 50 kDa ATF-6 (after 4 and 8 h of incubation in the presence of tunicamycin), compared to those of S cells. To verify this finding, we transfected S cells with a plasmid to induce GRP78/BiP transcription/translation and downregulation of CHOP expression after incubation with tunicamycin was observed. Transfected S cells behaved similarly to R and T cells but differently from untransfected S cells in monitoring cell cycle changes under the influence of tunicamycin. Thus, we can conclude that an increased level of GRP78/BiP is responsible for the ability of R and T cells to survive repeated passage in media containing 0.1 µM tunicamycin.