JOURNAL OF CELLULAR PHYSIOLOGY 150:632-639 (1992)
Susceptibility to Verotoxin as a Function of the Cell Cycle ANITA PUDYMAITIS AND CLIFFORD A. LINGWOOD* Department of Microbiology (A.P.,C.A.L.), and Biochemistry (C.A.L.), The Hospital for Sick Children, Toronto, Ontario M 5 G 1 X8, and the Departments of Clinical Biochemistry (A.P.,C.A.L.), Biochemistry (C.A.L.), and Microbiology (C.A.L.), University of Toronto, Toronto, Ontario, Canada Infection with Verotoxin producing fscherichia coli IVTEC) has been implicated in hemolytic uremic syndrome, the leading cause of pediatric renal failure. Verotoxin (VT) binds to globotriaosylceramide (Gala1-4GalP1-4GlcCer Gb,) in susceptible cells. Gb, is required for cytotoxicity and toxin-resistant cells deficient in Gb, can be sensitized to VT cytotoxicity by incorporation of exogenous Gb, into the cells. However, the absolute Gb, content of cell lines does not necessarily correspond directly with the degree of sensitivity to VT. The present study demonstrates that susceptibility to VT is a function of cell growth and that stationary phase cells are resistant to VT. Using chemically synchronized Vero cells, we have also found a tenfold difference in susceptibility to VT during the cell cycle. Our experiments define a maximal sensitivity “window” of 1-2 hours from the G1/S boundary. This corresponds to increased VT binding without change in overall Gb, content. Cell surface labelling indicated that cyclic turnover and exposure of Gb, may be the critical parameter in determining VT sensitivity. Such changes during the cell cycle may also be of relevance in vivo in determining toxin pathology during VTEC infections and the physiology of plasma membrane
Verotoxins (VT) originally described by Konowalchuk (Konowalchuk et al., 1977), are a family of Escherichia coli derived toxins (also referred to a s Shigalike toxins), which have been implicated in the etiology of hemolytic uremic syndrome (HUS) (Karmali et al., 1983; Karmali et al., 1985b1, the leading cause of pediatric renal failure. VT1, VT2, and SLTII are distinct but homologous toxins that bind to the glycolipid globotriaosyl ceramide (galal-4galpl-4glc ceramide-Gb,) (DeGrandis et al., 1989). Although many advances have recently been made in understanding the biology of VT at the molecular level, relatively little is understood regarding the precise role of VT in the pathogenesis of HUS and related syndromes. Recent reports have suggested that renal vascular endothelial cells may be the principle target in HUS. VT1 administered intravenously to rabbits produces microangiopathic changes with abnormal endothelial morphology (Zoja et al., submitted), resembling the pathologic findings in clinical HUS (Richardson et al., 1988). In addition, Obrig et al. (1988) have reported that purified VT is cytotoxic to human endothelial cells in tissue culture. Cell susceptibility to VT correlates with the presence (though not the level) of Gb, (Lingwood et al., 1987; Cohen et al., 1990). Incorporation of Gb, into (Waddell et al., 1990), or induction of Gb, synthesis in (Sandvig et al., 1991) receptor negative cultured cells induces VT sensitivity. High levels of Gb, have been found in human kidney (Boyd and Lingwood, 19891, particularly in the cortex, further implicating VT in the observed renal pathology of HUS. However, 0 1992 WILEY-LISS, INC.
the concentration of renal Gb, was not found to correlate with the age related incidence of HUS following VTEC infection. VT has been found to have a more toxic effect on young, dividing as compared to established, confluent endothelial cells (Obrig e t al., 1987). It has been proposed that this difference is a n important mechanism in VT-induced HUS. The reasons for the observed differences in sensitivity to VT as a function of growth have been speculative. Both confluent and non-confluent cells bind VT to their surfaces, hence active cell division may be a prerequisite for the expression of the maximum toxin sensitivity of target cells. Since sensitivity to VT was found to be a function of endothelial cell growth in culture, a n analysis of VT1 sensitivity during the cell cycle of Vero cells, which are the standard cells used for detecting this toxin (Karmali et al., 1985131, was performed.
MATERIALS AND METHODS Glycolipid standards lactosylceramide, Gb,, and Gb, were purified from human kidney by silicic acid chromatography (Strasberg et al., 1989); Galactosylceramide (GalCer), glucosylceramide (GlcCer) and digalactosyldiacylglycerol (DGDG) were obtained from Matreya (Pa); thymidine was obtained from Sigma and
Received July 17, 1991; accepted October 14,1991 *To whom reprint requestsicorrespondenceshould be addressed.
SUSCEPTIBILITY TO VEROTOXIN AS A FUNCTION OF THE CELL CYCLE
633
Extraction and high pressure liquid chromatography (HPLC) quantitation of Gb, during different stages of the cell cycle Cells were TdR synchronized in 75 cm2 flasks. At Cell culture different stages of the cell cycle, cells were detached The Vero cell line used in these studies originated with 1%trypsin, washed and resuspended in PBS from Green African monkey kidney tissue and was pH 7.4, and counted. maintained in Dulbecco’s modified essential medium The glycolipids were then extracted in 20 volumes supplemented with penicillin and streptomycin, vita- of chloroform/methanol 2: 1 (v/v). After filtering the mins, L-glutamine, and 5% fetal calf serum. The Vero residue through glass wool, a Folch extraction for tocell verotoxin-resistant cell line (VRP) was a vero cell tal lipid was performed, and 10 nmol of galactosyl diclone selected for its ability to grow in the presence of glyceride internal standard was added to each sample. VT2 and found to lack Gb, due to a loss of a-galactosyl Glycolipids were separated by silicic acid chromatransferase activity (Pudymaitis et al., 1991). tography (Lingwood et al., 1987). Glycolipids eluted with acetonelmethanol 9:l were benzoylated (Boyd and Cell synchronization Lingwood, 1989). Cells were synchronized primarily by double thymiSamples were dissolved in pyridine containing 10% dine (TdR) blockade (Thomas and Lingwood, 1975). benzoyl chloride and maintained overnight a t 37°C. Logarithmically growing Vero cells were incubated The reaction mixture was dried under N2 to remove the with thymidine (2 x lo-, M) for a period equal to the pyridine, and the benzoylated glycolipids were dismean generation time of 24 hours. Cells were then solved in hexane and washed with alkaline methanol. washed with PBS and re-incubated with fresh media for The clean upper hexane phase was applied to a silica a period greater than S phase (> 8 hours). A second gel 60 column and eluted with 5% isopropanol in hexthymidine block was then introduced for a further 16 ane, and then with methanol/benzene 9:l. Dried Samhours. Cells were washed and fresh media was added ples were resuspended in carbon tetrachloride and apwhen growth was initiated a t the start of the S phase. plied to a normal phase silica HPLC column. The G2 was a t 9 hours and mitosis occurred between 12 and column was eluted with a linear 0-15% isopropanol in 15 hours after release. hexane gradient. Peak heights were integrated relative Alternatively, cells were synchronized by subculture to the internal standard and Gb, was defined by comfrom confluent “contact inhibited” cultures. For these parison with a known standard (Strasberg et al., 1989). cells, there was a n approximate 12 hour lag before cell cycle entry. S was observed 13-20 hours after splitting 14C-galactoseincorporation and mitosis occurred over 2 hours after 24 hours 14C-Galactose (15 mCi/mole) was added to synchroculture. Synchronous growth was monitored by measurement nized cells for a 2 hour pulse (2 pCi/25 cm2 flask). Glyof rate of incorporation of ,H-thymidine into DNA and colipids were then extracted as described above and increase in cell number (Thomas and Lingwood, 1975). separated by TLC (chloroformimethanollwater Due to the requirement for a significant number of cells [65;25:4, v/v/v]). After autoradiography, relative labelfor biochemical assay a t the initial time points, a sec- ling of Gb, was calculated. ond round of synchronous division was not usually observed. Fluorescein isothiocyanate labelling of VT Approximately 50 pg of purified VT2 (Head et al., Verocytotoxin assay 1988) was dialyzed against borate buffer (50 mM, pH Verotoxin (VT1) was titrated using either twofold or 9.0) overnight. Fluorescein isothiocyanate (FITC) (1 tenfold dilutions onto a monolayer of cells. One 50% mg/ml) was added to the dialyzed toxin and shaken cytotoxicity dose (CD50 unit) was defined as the well. The mixture was allowed to react in the dark for 2 amount of VT1 that caused a cytotoxic effect in 50% of a hours at 4°C and then dialyzed against PBS (Law and cell monolayer after 3 days culture (Karmali et al., Lingwood, 1985). The fluorescein-conjugated VT 1985a). (FITC-VT) was then aliquoted and stored a t -70°C. The FITC-VT was titrated for cytotoxic effect upon Vero Glycolipid extraction of cultured cells cells by the standard cytotoxicity assay. The FITC-VT Synchronized cells (approximately 107/plate) were was also visualized by UV light illumination after SDSwashed and resuspended in 1ml of phosphate-buffered PAGE. saline, extracted in 20 volumes of chloroform/methanol, 2:1(v/v), and filtered through glass wool. The filtered FITC-VT labelling of cells residue was partitioned against water and the lower Vero cells were synchronized on eight well chamber phase was collected (Folch et al., 1957). Where indicated, the lower phase was then saponified in 1 N slides (Lab-Tek Products, Naperville, Ill.) (approxiNaOH in methanol (2-5 mg lipid/ml) for 2 hours a t mately 5 x lo4 cells per well). At different stages of the 37°C and the solution was neutralized with 1 N HC1. cell cycle, cells were labelled with FITC-VT (0.5 pg in The glycolipids were recovered by Folch extraction and 100 +L of 2% BSA in PBS) for 1hour a t 4°C in the dark run through glass wool to remove any residual salt. to minimize photobleaching. Following gentle washing, After repartitioning, the lower phase was dried and the the slides were fixed in 4% paraformaldehyde mounted residue resuspended in chloroform/methanol, 2:l (v/v). in a 75% by weight glycerol solution in 10 mM TBS
{,H}-thymidine (2 Ci/mM) was from ICN.VT1 and VT2 were prepared as previously described (Petric et al., 1987; Head e t al., 1988).
634
PUDYMAITIS AND LINGWOOD
r
TABLE la. Verotoxin sensitivity as a function of growth Titres asynchronous cells 24 hours 72 hours
sparse (log phase)
confluent (stationary phase)
64 32
Susceptibility to Verotoxin as a Function of the Cell Cycle ANITA PUDYMAITIS AND CLIFFORD A. LINGWOOD* Department of Microbiology (A.P.,C.A.L.), and Biochemistry (C.A.L.), The Hospital for Sick Children, Toronto, Ontario M 5 G 1 X8, and the Departments of Clinical Biochemistry (A.P.,C.A.L.), Biochemistry (C.A.L.), and Microbiology (C.A.L.), University of Toronto, Toronto, Ontario, Canada Infection with Verotoxin producing fscherichia coli IVTEC) has been implicated in hemolytic uremic syndrome, the leading cause of pediatric renal failure. Verotoxin (VT) binds to globotriaosylceramide (Gala1-4GalP1-4GlcCer Gb,) in susceptible cells. Gb, is required for cytotoxicity and toxin-resistant cells deficient in Gb, can be sensitized to VT cytotoxicity by incorporation of exogenous Gb, into the cells. However, the absolute Gb, content of cell lines does not necessarily correspond directly with the degree of sensitivity to VT. The present study demonstrates that susceptibility to VT is a function of cell growth and that stationary phase cells are resistant to VT. Using chemically synchronized Vero cells, we have also found a tenfold difference in susceptibility to VT during the cell cycle. Our experiments define a maximal sensitivity “window” of 1-2 hours from the G1/S boundary. This corresponds to increased VT binding without change in overall Gb, content. Cell surface labelling indicated that cyclic turnover and exposure of Gb, may be the critical parameter in determining VT sensitivity. Such changes during the cell cycle may also be of relevance in vivo in determining toxin pathology during VTEC infections and the physiology of plasma membrane
Verotoxins (VT) originally described by Konowalchuk (Konowalchuk et al., 1977), are a family of Escherichia coli derived toxins (also referred to a s Shigalike toxins), which have been implicated in the etiology of hemolytic uremic syndrome (HUS) (Karmali et al., 1983; Karmali et al., 1985b1, the leading cause of pediatric renal failure. VT1, VT2, and SLTII are distinct but homologous toxins that bind to the glycolipid globotriaosyl ceramide (galal-4galpl-4glc ceramide-Gb,) (DeGrandis et al., 1989). Although many advances have recently been made in understanding the biology of VT at the molecular level, relatively little is understood regarding the precise role of VT in the pathogenesis of HUS and related syndromes. Recent reports have suggested that renal vascular endothelial cells may be the principle target in HUS. VT1 administered intravenously to rabbits produces microangiopathic changes with abnormal endothelial morphology (Zoja et al., submitted), resembling the pathologic findings in clinical HUS (Richardson et al., 1988). In addition, Obrig et al. (1988) have reported that purified VT is cytotoxic to human endothelial cells in tissue culture. Cell susceptibility to VT correlates with the presence (though not the level) of Gb, (Lingwood et al., 1987; Cohen et al., 1990). Incorporation of Gb, into (Waddell et al., 1990), or induction of Gb, synthesis in (Sandvig et al., 1991) receptor negative cultured cells induces VT sensitivity. High levels of Gb, have been found in human kidney (Boyd and Lingwood, 19891, particularly in the cortex, further implicating VT in the observed renal pathology of HUS. However, 0 1992 WILEY-LISS, INC.
the concentration of renal Gb, was not found to correlate with the age related incidence of HUS following VTEC infection. VT has been found to have a more toxic effect on young, dividing as compared to established, confluent endothelial cells (Obrig e t al., 1987). It has been proposed that this difference is a n important mechanism in VT-induced HUS. The reasons for the observed differences in sensitivity to VT as a function of growth have been speculative. Both confluent and non-confluent cells bind VT to their surfaces, hence active cell division may be a prerequisite for the expression of the maximum toxin sensitivity of target cells. Since sensitivity to VT was found to be a function of endothelial cell growth in culture, a n analysis of VT1 sensitivity during the cell cycle of Vero cells, which are the standard cells used for detecting this toxin (Karmali et al., 1985131, was performed.
MATERIALS AND METHODS Glycolipid standards lactosylceramide, Gb,, and Gb, were purified from human kidney by silicic acid chromatography (Strasberg et al., 1989); Galactosylceramide (GalCer), glucosylceramide (GlcCer) and digalactosyldiacylglycerol (DGDG) were obtained from Matreya (Pa); thymidine was obtained from Sigma and
Received July 17, 1991; accepted October 14,1991 *To whom reprint requestsicorrespondenceshould be addressed.
SUSCEPTIBILITY TO VEROTOXIN AS A FUNCTION OF THE CELL CYCLE
633
Extraction and high pressure liquid chromatography (HPLC) quantitation of Gb, during different stages of the cell cycle Cells were TdR synchronized in 75 cm2 flasks. At Cell culture different stages of the cell cycle, cells were detached The Vero cell line used in these studies originated with 1%trypsin, washed and resuspended in PBS from Green African monkey kidney tissue and was pH 7.4, and counted. maintained in Dulbecco’s modified essential medium The glycolipids were then extracted in 20 volumes supplemented with penicillin and streptomycin, vita- of chloroform/methanol 2: 1 (v/v). After filtering the mins, L-glutamine, and 5% fetal calf serum. The Vero residue through glass wool, a Folch extraction for tocell verotoxin-resistant cell line (VRP) was a vero cell tal lipid was performed, and 10 nmol of galactosyl diclone selected for its ability to grow in the presence of glyceride internal standard was added to each sample. VT2 and found to lack Gb, due to a loss of a-galactosyl Glycolipids were separated by silicic acid chromatransferase activity (Pudymaitis et al., 1991). tography (Lingwood et al., 1987). Glycolipids eluted with acetonelmethanol 9:l were benzoylated (Boyd and Cell synchronization Lingwood, 1989). Cells were synchronized primarily by double thymiSamples were dissolved in pyridine containing 10% dine (TdR) blockade (Thomas and Lingwood, 1975). benzoyl chloride and maintained overnight a t 37°C. Logarithmically growing Vero cells were incubated The reaction mixture was dried under N2 to remove the with thymidine (2 x lo-, M) for a period equal to the pyridine, and the benzoylated glycolipids were dismean generation time of 24 hours. Cells were then solved in hexane and washed with alkaline methanol. washed with PBS and re-incubated with fresh media for The clean upper hexane phase was applied to a silica a period greater than S phase (> 8 hours). A second gel 60 column and eluted with 5% isopropanol in hexthymidine block was then introduced for a further 16 ane, and then with methanol/benzene 9:l. Dried Samhours. Cells were washed and fresh media was added ples were resuspended in carbon tetrachloride and apwhen growth was initiated a t the start of the S phase. plied to a normal phase silica HPLC column. The G2 was a t 9 hours and mitosis occurred between 12 and column was eluted with a linear 0-15% isopropanol in 15 hours after release. hexane gradient. Peak heights were integrated relative Alternatively, cells were synchronized by subculture to the internal standard and Gb, was defined by comfrom confluent “contact inhibited” cultures. For these parison with a known standard (Strasberg et al., 1989). cells, there was a n approximate 12 hour lag before cell cycle entry. S was observed 13-20 hours after splitting 14C-galactoseincorporation and mitosis occurred over 2 hours after 24 hours 14C-Galactose (15 mCi/mole) was added to synchroculture. Synchronous growth was monitored by measurement nized cells for a 2 hour pulse (2 pCi/25 cm2 flask). Glyof rate of incorporation of ,H-thymidine into DNA and colipids were then extracted as described above and increase in cell number (Thomas and Lingwood, 1975). separated by TLC (chloroformimethanollwater Due to the requirement for a significant number of cells [65;25:4, v/v/v]). After autoradiography, relative labelfor biochemical assay a t the initial time points, a sec- ling of Gb, was calculated. ond round of synchronous division was not usually observed. Fluorescein isothiocyanate labelling of VT Approximately 50 pg of purified VT2 (Head et al., Verocytotoxin assay 1988) was dialyzed against borate buffer (50 mM, pH Verotoxin (VT1) was titrated using either twofold or 9.0) overnight. Fluorescein isothiocyanate (FITC) (1 tenfold dilutions onto a monolayer of cells. One 50% mg/ml) was added to the dialyzed toxin and shaken cytotoxicity dose (CD50 unit) was defined as the well. The mixture was allowed to react in the dark for 2 amount of VT1 that caused a cytotoxic effect in 50% of a hours at 4°C and then dialyzed against PBS (Law and cell monolayer after 3 days culture (Karmali et al., Lingwood, 1985). The fluorescein-conjugated VT 1985a). (FITC-VT) was then aliquoted and stored a t -70°C. The FITC-VT was titrated for cytotoxic effect upon Vero Glycolipid extraction of cultured cells cells by the standard cytotoxicity assay. The FITC-VT Synchronized cells (approximately 107/plate) were was also visualized by UV light illumination after SDSwashed and resuspended in 1ml of phosphate-buffered PAGE. saline, extracted in 20 volumes of chloroform/methanol, 2:1(v/v), and filtered through glass wool. The filtered FITC-VT labelling of cells residue was partitioned against water and the lower Vero cells were synchronized on eight well chamber phase was collected (Folch et al., 1957). Where indicated, the lower phase was then saponified in 1 N slides (Lab-Tek Products, Naperville, Ill.) (approxiNaOH in methanol (2-5 mg lipid/ml) for 2 hours a t mately 5 x lo4 cells per well). At different stages of the 37°C and the solution was neutralized with 1 N HC1. cell cycle, cells were labelled with FITC-VT (0.5 pg in The glycolipids were recovered by Folch extraction and 100 +L of 2% BSA in PBS) for 1hour a t 4°C in the dark run through glass wool to remove any residual salt. to minimize photobleaching. Following gentle washing, After repartitioning, the lower phase was dried and the the slides were fixed in 4% paraformaldehyde mounted residue resuspended in chloroform/methanol, 2:l (v/v). in a 75% by weight glycerol solution in 10 mM TBS
{,H}-thymidine (2 Ci/mM) was from ICN.VT1 and VT2 were prepared as previously described (Petric et al., 1987; Head e t al., 1988).
634
PUDYMAITIS AND LINGWOOD
r
TABLE la. Verotoxin sensitivity as a function of growth Titres asynchronous cells 24 hours 72 hours
sparse (log phase)
confluent (stationary phase)
64 32
