Diallyl disulfide inhibits p34cdc2 kinase activity through changes in complex formation and phosphorylation

Lyn M. Knowles and John A. Milner1

Graduate Program in Nutrition and the Nutrition Department, 126 Henderson Building South, The Pennsylvania State University, University Park, PA 16802, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous studies from our laboratory demonstrated that diallyl disulfide (DADS), an oil-soluble allyl sulfur compound found in processed garlic, markedly suppressed p34cdc2 kinase activity and induced a G2/M phase arrest in cultured human colon tumor (HCT-15) cells. The present studies reveal that suppression of p34cdc2 kinase activity by DADS does not result from direct interactions with the protein, but through changes in factors influencing the formation and conversion of the enzyme to its active form. Flow cytometric analyses showed that the increased proportion of cells in the G2/M phase following DADS treatment was accompanied by an increase in cyclin B1 protein expression. A temporal and dose-dependent response in cyclin B1 expression was observed in cells treated with DADS. Western blot analysis revealed that 50 µM DADS did not influence the quantity of p34cdc2 protein expressed, but did decrease the amount associated with cyclin B1 by 26% (P < 0.05). Exposure of unsynchronized cells to 25 or 50 µM DADS caused a trend towards increased p34cdc2 hyperphosphorylation (17 and 22%, respectively). Exposure of synchronized cells to 100 µM DADS increased p34cdc2 hyperphosphorylation by 15% (P < 0.05). Consistent with its ability to slightly increase the quantity of hyperphosphorylated p34cdc2, DADS, 25 or 50 µM, decreased cdc25C protein expression by 23 and 46%, respectively (P < 0.05). The present studies suggest that the ability of DADS to inhibit p34cdc2 kinase activation occurs because of decreased p34cdc2/cyclin B1 complex formation and modest p34cdc2 hyperphosphorylation.

Abbreviations: DADS, diallyl disulfide.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Garlic (Allium sativum) has historically been used to alleviate a wide variety of health problems (1). Recently, laboratory investigations have ascribed antimicrobial, antimutagenic, antithrombic and anticancer properties to garlic and its associated compounds (24). Evidence of garlic's anticarcinogenic role comes from studies that have correlated an increased consumption of garlic with a reduction in the development of a variety of cancers (57). Furthermore, consumption of organic and water extracts of garlic by experimental animals inhibits the initiation of chemically induced cancers through the suppression of carcinogen formation and bioactivation (812).

In addition to modifying processes involved in the initiation of carcinogenesis, specific allyl organosulfur compounds within processed garlic have been shown to suppress the proliferation of cultured tumor cells (1315). Previous studies from our laboratory demonstrated that the oil-soluble compound diallyl disulfide (DADS) was more effective in inhibiting the growth of human colon, lung and skin tumor cells than the non-allyl dipropyl disulfide or water-soluble S-allyl cysteine (13). The ability of DADS to suppress proliferation correlated with a decrease in cellular thiols, an increase in intracellular calcium, and the induction of apoptosis (1315). More recent observations demonstrate that the antiproliferative effects of DADS are associated with a suppression in cell division through a G2/M phase arrest (16).

Activation of the p34cdc2 kinase complex is known to modulate the progression of cells from the G2 into the M phase of the cell cycle by promoting chromosome condensation, cytoskeletal reorganization and nuclear envelope breakdown (17,18). Recent studies from our laboratory revealed that DADS caused a marked suppression in p34cdc2 kinase activity (16). Central to p34cdc2 kinase activation is the association of the p34cdc2 catalytic subunit with the cyclin B1 regulatory unit (19). Cyclin B1 synthesis and accumulation in the S phase are known to regulate p34cdc2 kinase activity (17,19,20). Degradation of cyclin B1 following anaphase by ubiquitin-dependent proteolysis is necessary for inactivation of the complex, completion of mitosis and progression of the cell into the G1 phase (17,21).

Activation of p34cdc2 kinase is also controlled by the phosphorylation and subsequent dephosphorylation of the cyclin-dependent p34cdc2 protein kinase (19). p34cdc2 kinase is initially phosphorylated on Thr14, Tyr15 and Thr161 to form an inactive hyperphosphorylated p34cdc2 kinase complex (19) and activated by the removal of phosphate groups from Thr14 and Tyr15 (17,19). Inhibition of cdc25C phosphatase activity is recognized to prevent the conversion of the inactive hyperphosphorylated form of p34cdc2 kinase to its active state (22,23).

The present studies were designed to examine plausible mechanisms by which DADS suppresses p34cdc2 kinase activity. Specific studies examined whether changes in cyclin B1, p34cdc2 or cdc25C protein expression explained the observed decrease in p34cdc2 kinase activity following DADS exposure. Additional studies examined the impact of DADS on the phosphorylation status and proper formation of the p34cdc2/cyclin B1 complex.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
The human colon tumor cell line, HCT-15, was purchased from American Type Culture Collection (Rockville, MD). DADS was purchased from Fluka Chemika (Ronkonkoma, NY). SignaTECT cdc2 kinase assay system was obtained from Promega (Madison, WI). [{gamma}-32P]ATP (sp. act. >7000 Ci/mmol) was purchased from ICN Pharmaceuticals (Irvine, CA). FITC-conjugated anti-cyclin B1 was purchased from Pharmingen (San Diego, CA). Monoclonal anti-cyclin B1, anti-p34cdc2, anti-p34cdc2 agarose conjugate and polyclonal anti-cdc25C antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase-conjugated goat anti-mouse IgG was obtained from Pierce (Rockford, IL) and peroxidase-labeled goat anti-rabbit IgG was purchased from Gibco BRL (Grand Island, NY). All other chemicals were obtained from Sigma (St Louis, MO).

Culture conditions
HCT-15 cells were plated in 150 cm2 tissue culture flasks and incubated in RPMI 1640 medium (pH 7.2; Sigma) supplemented with 10% fetal bovine serum (FBS; Gibco BRL), 1% penicillin/streptomycin (10 000 U/ml penicillin and 10 mg/ml streptomycin) and 1 µg/ml insulin (15). Cells were plated at 4x103/cm2 for 48 h prior to treatment. Cells were grown under a humidified, 5% CO2 atmosphere at 37°C. DADS was dissolved in dimethyl sulfoxide (DMSO) prior to addition to cultures. Control cultures were treated with DMSO. The maximum quantity of DMSO added to the medium in these studies was 0.01%.

Cell proliferation
At specified incubation times, as indicated below, cells were washed with cold PBS (pH 7.2), harvested by trypsinization (0.025% trypsin-EDTA), rinsed with RPMI 1640 containing FBS to deactivate the trypsin and centrifuged at 500 g. Cells were resuspended in RPMI 1640 and viable cells were counted on a hemocytometer using trypan blue exclusion.

Cell synchronization and analysis of p34cdc2 kinase activity
HCT-15 cells were synchronized according to the double thymidine block method of Stein and Stein (24). Synchronized cells were harvested 6 h after the removal of the block to obtain maximum p34cdc2 kinase activity as determined previously (16) and lysed in a buffer containing 50 mM Tris–HCl (pH 7.4), 250 mM NaCl, 1 mM EDTA, 50 mM NaF, 50 µg/ml phenylmethylsulfonyl fluoride (PMSF), 2.2 µg/ml aprotinin and 0.7 µg/ml pepstatin A for 10 min, sonified and centrifuged at 100 000 g for 1 h at 4°C. The protein content of the resulting cell extract was determined using a standard kit (Pierce, Rockford, IL). In these studies, control cell extracts were incubated with various concentrations of DADS for 5 min prior to the assessment of p34cdc2 kinase activity as described previously (16).

Flow cytometric analysis of cyclin B1 and cellular DNA
HCT-15 cells were harvested at various times over 24 h, rinsed in cold PBS, fixed in 75% ethanol, and stored at –20°C for subsequent cell cycle analysis. Fixed cells were centrifuged at 500 g to remove the fixing agent, rinsed in a wash buffer of PBS supplemented with 1% FBS, centrifuged and incubated on ice in PBS containing 0.25% Triton X-100 for 5 min. The suspensions were then centrifuged, rinsed with wash buffer prior to antibody addition. Cell pellets were incubated in the dark for 30 min with FITC-conjugated cyclin B1 antibody. Following centrifugation to remove the antibody, cellular DNA was stained by the addition of PBS containing 10 µg/ml propidium iodide. Samples were incubated in the dark at 4°C for at least 10 min prior to analysis. Both protein expression and the percentage of cells in the G1, S and G2/M phases of the cell cycle were determined using a Coulter XL-MCL tabletop cytometer. From each culture harvested, the DNA of 10 000 cells was analyzed.

Cyclin B1, p34cdc2 and cdc25C western blots
HCT-15 cells were harvested, rinsed twice with cold PBS and incubated for 10 min in lysis buffer composed of either 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% igepal, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM dithiothreitol, 2 mM EDTA, 50 µg/ml PMSF, 2.2 µg/ml aprotinin, 0.7 µg/ml pepstatin A for analysis of cyclin B1, p34cdc2 and cdc25C protein levels or in PBS (pH 7.4) containing 1% igepal, 10 mM NaF, 1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, 50 µg/ml PMSF, 2.2 µg/ml aprotinin, 0.7 µg/ml pepstatin A for determination of p34cdc2 phosphorylation status. Samples were sonified, centrifuged and analyzed for protein content as described above. An aliquot of the cell extract was then subjected to electrophoresis on a 10% (cyclin B1 and p34cdc2), 12% (cdc25C) or 18% (p34cdc2 phosphorylation) SDS–acrylamide gel, transferred onto a nitrocellulose membrane and probed with anti-cyclin B1, p34cdc2 or cdc25C as the primary antibody and anti-mouse or anti-rabbit IgG conjugated peroxidase as a secondary antibody. Bands were detected by enhanced chemiluminescence (Amersham Life Science, Little Chalfont, UK). The relative density of each band was determined using NIH Imagine 1.61 software (NIH, Bethesda, MD).

p34cdc2 immunoprecipitation
HCT-15 cells were lysed according to the procedure used above in harvesting extracts for the analysis of p34cdc2 kinase activity. The correct assembly of p34cdc2 and cyclin B1 was measured following the conditions described by Barth and Kinzel (25). In brief, an aliquot of the cell extract was diluted to 0.5 ml with lysis buffer and incubated with p34cdc2 conjugated agarose at 4°C for 1 h on a rotator. Samples were pelleted by centrifugation for 5 min at 2000 r.p.m., washed three times in 1 ml lysis buffer, boiled in 2x SDS sample buffer for 10 min and loaded onto a 10% SDS–acrylamide gel. Following electrophoresis and transfer onto a nitrocellulose membrane, blots were probed with anti-cyclin B1 as the primary antibody and anti-mouse IgG conjugated peroxidase as a secondary antibody and detected as described above.

Statistical analysis
Analysis of variance (ANOVA) statistics were applied to the data using the Statistical Analysis System (SAS) for Windows version 6.12 (1996). Comparisons among treatments were calculated using Tukey's honestly significant difference test. Treatment mean differences with P < 0.05 were considered statistically different.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
DADS does not directly inhibit p34cdc2 kinase activity
To determine whether suppression of p34cdc2 kinase activity by DADS is the result of direct or indirect interactions, we examined its effect on the enzyme's activity when added to control cell lysates. At all concentrations examined, DADS (0, 1, 10, 50 or 100 µM) failed to directly suppress p34cdc2 activity (Figure 1Go). In fact, when concentrations were 50 µM or more there was an ~2-fold increase in activity.



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Fig. 1. Extracts of synchronized HCT-15 cells were examined for p34cdc2 kinase activity after the addition of increasing concentrations of DADS. DADS did not directly inhibit p34cdc2 activity when added to control cell lysates. The mean p34cdc2 kinase activity from control lysates was 75 fmol ATP/min/µg protein and was designated as 100% in the bar graph. Values are means ± SEM of four determinations per treatment performed in duplicate. Values not sharing a common letter (a, b) differ (P < 0.05).

 
Effect of DADS on cyclin B1 protein expression
Western blot analysis of cyclin B1 was used to determine whether decreased protein expression could explain the suppression of p34cdc2 kinase activity by DADS. Cyclin B1 protein expression was examined after a 12 h exposure to DADS since our previous studies showed an increase in the proportion of cells arrested in the G2/M phase (16). Treatment with 50 µM DADS for 12 h resulted in a 2-fold increase in cyclin B1 expression (Figure 2Go). Figure 3Go demonstrates the temporal effects of DADS (0, 25 or 50 µM) on cyclin B1 protein expression and the distribution of cells within the various phases of the cell cycle. Flow cytometric analyses revealed that cyclin B1 protein was enhanced within 4 h after exposure to 25 or 50 µM DADS (Figure 3AGo). Whereas cyclin B1 expression in cells exposed to 25 µM DADS returned toward control values by 8 h, expression remained elevated after 8 h in cells treated with 50 µM DADS (Figure 3AGo). Cyclin B1 expression after a 24 h exposure to either 25 or 50 µM DADS was not different from controls.



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Fig. 2. The effect of DADS (50 µM) on cyclin B1 expression in HCT-15 cells. HCT-15 cell lysates were separated by SDS–PAGE and probed for cyclin B1 expression. The upper panel shows a representative immunoblot. The mean cyclin B1 expression from control lysates was 352 arbitrary units and was designated as 100% in the graph. Each bar represents the mean ± SEM cyclin B1 protein expression 12 h after treatment for three determinations per treatment performed in duplicate. Values not sharing a common letter (a, b) differ (P < 0.05). DADS treatment significantly increased cyclin B1 expression compared with controls.

 


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Fig. 3. (A) The impact of increasing concentrations of DADS on cyclin B1 protein expression. DADS treatment significantly increased cyclin B1 expression in a dose- and time-dependent manner. Values are means ± SEM of three determinations per treatment. The maximum SEM observed for all measurements was ±3.3%. (B) The influence of increasing concentrations of DADS on the percentage of cells in the G2/M phase. Values are means ± SEM of three determinations per treatment. The maximum SEM observed for all measurements was ±4.8%.

 
Cell cycle analysis revealed that the proportion of cells in the G2/M phase increased after exposure to 25 or 50 µM DADS (Figure 3BGo). The equation explaining the relationship between G2/M cell cycle arrest and cyclin B1 expression was y = 0.564x – 39.49 (r = 0.821). The ability of 25 µM DADS to arrest cells in the G2/M phase persisted beyond its ability to increase cyclin B1 expression (Figure 3A and BGo).

Effect of DADS on p34cdc2 protein expression
To determine whether changes in p34cdc2 kinase activity were reflected by alterations in p34cdc2 protein levels, we examined the influence of DADS on its expression using western blot analysis. HCT-15 cell lysates were prepared in the absence of phosphatase inhibitors and were found to express a single p34cdc2 isoform when immunoblots were probed with anti-p34cdc2. Similar levels of p34cdc2 were expressed in cultures grown in the presence or absence of DADS (0, 25 or 50 µM; Figure 4Go).



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Fig. 4. The effect of DADS (25 and 50 µM) on p34cdc2 expression in HCT-15 cells. Cell lysates were separated by SDS–PAGE and probed for p34cdc2 expression. The mean p34cdc2 expression from control lysates was 823 arbitrary units and was designated as 100% in the graph. Each bar represents the mean p34cdc2 protein expression ± SEM of three determinations per treatment performed in duplicate at 12 h after treatment, except for one control with one replicate. Significant differences in p34cdc2 expression were not observed following exposure to DADS.

 
DADS decreases p34cdc2/cyclin B1 complex formation
Since the activation of p34cdc2 kinase also requires the interaction of cyclin B1 with p34cdc2, we assessed the impact of DADS on the expression of the complex. The influence of a 12 h exposure to DADS (0, 25 or 50 µM) on p34cdc2/cyclin B1 complex formation is illustrated in Figure 5Go. While no significant change in complex expression occurred in cells exposed to 25 µM DADS, 50 µM decreased levels by 26% compared with control values (P < 0.05).



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Fig. 5. The impact of DADS (25 and 50 µM) on the formation of the p34cdc2/cyclin B1 complex. Cell lysates were immunoprecipitated with p34cdc2 agarose-conjugated antibody, separated by SDS–PAGE and probed for cyclin B1 expression. The upper panel shows a representative immunoblot. The mean cyclin B1 expression from control lysates was 689 arbitrary units and was designated as 100% in the graph. Each bar represents mean cyclin B1 protein expression ± SEM of three determinations per treatment performed in quadruplicate at 12 h after treatment, except for one 50 µM determination which had only three replicates. Significant differences in p34cdc2/cyclin B1 complex expression were observed following exposure to 50 µM DADS.

 
DADS induces p34cdc2 hyperphosphorylation
To ascertain whether changes in p34cdc2 phosphoryation also contribute to the suppression in p34cdc2 kinase activity by DADS, the phosphorylation status of p34cdc2 was assessed by western blot analysis. Phosphorylation of p34cdc2 is known to increase its molecular mass causing it to migrate on SDS–polyacrylamide gels as three distinct isoforms (26). These isoforms represent the hypophosphorylated (lower) and the hyperphosphorylated (middle and upper) forms of the protein. To maximize the recovery of phosphorylated p34cdc2, HCT-15 cells were lysed in the presence of phosphatase inhibitors whereby all three isoforms were isolated (Figure 6A and BGo). Exposure of cells to 25 or 50 µM DADS for 12 h did not significantly alter p34cdc2 phosphorylation status compared with controls (Figure 6AGo). Although a significant change in p34cdc2 phosphorylation was not observed, a trend towards an increase (17 and 22%) in hyperphosphorylated and a decrease (10 and 14%) in the hypophosphorylated isoform was observed when values were expressed as a percentage of total p34cdc2 expression in cultures treated with 25 or 50 µM DADS, respectively. To maximize the ability to detect changes in p34cdc2 phosphorylation, we decided to analyze the effect of 100 µM DADS on its phosphorylation in synchronized HCT-15 cells. Since previous studies revealed that p34cdc2 kinase activity was markedly reduced by 4 h in synchronized cultures, this time was chosen for this study (16). In the present study, a 4 h exposure to 100 µM DADS resulted in a 15% increase in the percentage of hyperphosphorylated p34cdc2 and a 15% decrease in the hypophosphorylated isoform compared with control values (P < 0.05; Figure 6BGo).



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Fig. 6. The effect of DADS on p34cdc2 phosphorylation status. Lysates were separated by SDS–PAGE and probed for p34cdc2 phosphorylation. (A) DADS (25 or 50 µM) did not alter p34cdc2 phosphorylation in unsynchronized cultures. The mean unphosphorylated and phosphorylated p34cdc2 expressions from control lysates were 414 and 291 arbitrary units, respectively, and are presented as percentage of total expression in the graph. Each bar represents p34cdc2 phosphorylation ± SEM of three determinations per treatment performed in quadruplicate at 12 h after treatment, except for two 50 µM determinations which had only three replicates. (B) DADS (100 µM) treatment significantly increased p34cdc2 phosphorylation in synchronized cultures. The mean unphosphorylated and phosphorylated p34cdc2 expressions from control lysates were 324 and 329 arbitrary units, respectively, and are presented as percentage of total expression in the graph. Each bar represents the mean p34cdc2 phosphorylation ± SEM of four determinations per treatment performed in quadruplicate at 12 h after treatment. Values within each phosphorylated form not sharing a common letter (a, b) differ (P < 0.05).

 
DADS suppresses cdc25C phosphatase expression
Western blot analysis was used to determine whether changes in cdc25C phosphatase expression could explain the ability of DADS to induce p34cdc2 hyperphosphoryation. Cell lysates were again prepared in the absence of phosphatase inhibitors and were found to express a single cdc25C isoform when immunoblots were probed with anti-cdc25C (Figure 7Go). Treatment with 25 or 50 µM DADS decreased 12 h cdc25C protein expression by 23 and 46%, respectively (P < 0.05; Figure 7Go).



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Fig. 7. The influence of DADS (25 and 50 µM) on cdc25C protein phosphatase expression in HCT-15 cells. Cell lysates were separated by SDS–PAGE and probed for cdc25C expression. The upper panel shows a representative immunoblot. The mean cdc25C expression from control lysates was 588 arbitrary units and was designated as 100% in the graph. Each bar represents the mean ± SEM cdc25C protein expression for three determinations per treatment performed in duplicate at 12 h after treatment Values not sharing a common letter (a, b) differ (P < 0.05). DADS treatment significantly decreased cdc25C expression compared with controls.

 

    Discussion
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 Abstract
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 Materials and methods
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 References
 
The present studies assist in clarifying the mechanism(s) through which DADS suppresses p34cdc2 kinase activity. Although DADS has been shown to directly suppress the activity of a number of enzymes by competitively inhibiting substrate–enzyme interactions (27,28), this is not the case for p34cdc2 kinase activity (Figure 1Go). In fact, a substantial increase in kinase activity occurred when DADS was added to cell lysates at 50 µM or more. The reason for this increase while unclear may relate to an unfolding of the complex and greater access to its active site. Our previous studies revealed that a decrease in p34cdc2 kinase activity caused by 50 µM DADS correlated with a suppression in HCT-15 proliferation (16). Since 50 µM DADS causes a marked suppression in p34cdc2 kinase activity in intact cells, the present response must be secondary to changes in cell signaling.

Indirect modification of p34cdc2 kinase activity is mediated through changes in either complex formation or its subsequent activation (17). Using western blot analysis, we demonstrated that p34cdc2/cyclin B1 complex expression decreased following exposure to DADS. Suppressions in p34cdc2 or cyclin B1 protein expression were not observed suggesting that DADS is not decreasing the synthesis of the proteins required for formation of the complex. Consequently, DADS may be altering complex expression either by decreasing p34cdc2 and cyclin B1 association or increasing their dissociation. Increased dissociation between p34cdc2 and cyclin B1 has been reported as a possible mechanism explaining depressed complex expression following abnormal elevations in intracellular calcium levels (29). Since increased intracellular calcium levels correlate with the ability of DADS to reduce HCT-15 tumor proliferation (13), increased complex dissociation may account for our observed decrease in p34cdc2/cyclin B1 complex expression.

Increased cyclin B1 protein expression during the G2/M transition has been demonstrated in a number of models (30,31). Using flow cytometric analysis, the present studies revealed that cyclin B1 expression increased in response to DADS and correlated with the block in G2/M progression. The elevation of G2/M cells persisted beyond the increase in protein expression suggesting that increased cyclin B1 levels may simply reflect the increased proportion of arrested cells. Considerable evidence points toward a role of active p34cdc2 kinase in phosphorylating cyclin B1 and targeting it for ubiquitin-dependent proteolysis (21,32). Suppressed p34cdc2 kinase activity could be preventing cyclin B1 degradation in the present studies.

Thr14 and Tyr15 are inhibitory residues whose phosphorylation provides a mechanism for the immediate inactivation of newly formed p34cdc2/cyclin B1 complexes (19,23). Removal of these residues activates p34cdc2 kinase (19,22,23). A number of antitumorigenic agents suppress p34cdc2 kinase activity by preventing the dephosphorylation of these residues (3336). The relatively small increase in the percentage of phosphorylated p34cdc2 kinase observed in these studies reveals that HCT-15 cell proliferation may be sensitive to subtle changes in phosphorylation or that this is not a major mechanism accounting for the depression in p34cdc2 kinase activity following DADS exposure. Analytical difficulties may have reduced our ability to detect changes in p34cdc2 phosphorylation in unsynchronized cultures. Nevertheless, even in synchronized cells the degree of hyperphosphorylation following DADS exposure was relatively modest. Additional studies are needed to determine if these changes might account for the temporal response in p34cdc2 kinase activity following DADS treatment (16).

In mammalian cells, activation of the p34cdc2 complex requires dephosphorylation of Thr14 and Tyr15 residues by a dual-specific protein phosphatase, cdc25C (23). In the present studies, p34cdc2 hyperphosphorylation was accompanied by a depletion in cdc25C phosphatase expression. Similar depressions in protein dephosphorylation by other allyl sulfur compounds have been reported (37,38). Specifically, Lee et al. (37) found increased smooth muscle cell p34cdc2 phosphorylation following treatment with S-allylmercaptocysteine. Suppressions in protein tyrosine phosphatase activity by ajoene also corresponded to an increase in the phosphorylation of a number of proteins (38). Consequently, alterations in p34cdc2 phosphorylation through suppression of cdc25C phosphatase activity may explain the observed growth inhibitory effects of a number of allyl sulfur compounds.

Based on the above results, we propose the following scheme as a possible explanation for DADS induced inactivation of p34cdc2 kinase (Figure 8Go). The present studies demonstrate that the ability of DADS to inhibit p34cdc2 kinase activity relates to a decrease in p34cdc2/cyclin B1 complex expression. In addition, these studies suggest that subtle increases in p34cdc2 hyperphosphorylation may accompany the decrease in complex expression. Thus, DADS may decrease the amount of the complex formed that is converted to the active enzyme.



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Fig. 8. Illustration of the p34cdc2 complex and the role of DADS in inhibiting kinase activation.

 
In summary, DADS probably modulates p34cdc2 kinase activity through changes in both complex formation and its subsequent activation. While DADS-mediated suppression of cdc25C likely accounts for increased p34cdc2 phosphorylation, increased cyclin B1 levels likely result from p34cdc2 kinase inactivation. The exact mechanism(s) governing decreased p34cdc2/cyclin B1 complex expression remain unknown since suppressions in p34cdc2 or cyclin B1 were not observed. Additional studies are needed to clarify the role of DADS in modifying p34cdc2/cyclin B1 complex expression.


    Notes
 
1 To whom correspondence should be addressed Email: jam14{at}psu.edu Back


    Acknowledgments
 
These studies were supported in part by a grant from the American Institute for Cancer Research. Some of these results were presented at Experimental Biology 98, San Francisco, CA, and have been published in abstract form [FASEB J., 12, 3814 (1998)].


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 3, 1999; revised February 9, 2000; accepted February 29, 2000.