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J. Biol. Chem., Vol. 276, Issue 41, 37815-37820, October 12, 2001

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Expression of p21^Waf1/Cip1 and Cyclin D1 Is Increased in Butyrate-resistant HeLa Cells^*

Anna Derjuga, Christina Richard^§, Milena Crosato, Paul S. Wright^¶, Lorraine Chalifour, Joe Valdez, Anna Barraso, Harry A. Crissman, Walter Nishioka^**, E. Morton Bradbury, and John P. H. Th'ng^§

From the  Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, Quebec, H3T 1E2, Canada, ^¶ Aventis Pharmaceutical Inc., Bridgewater, New Jersey 08807,  Los Alamos National Laboratories, New Mexico 87545, ^** Vical Inc., San Diego, California 92121, and ^§ Northwestern Ontario Regional Cancer Center, Thunder Bay, Ontario P7A 7T1, Canada

Received for publication, May 23, 2001, and in revised form, July 18, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Sodium butyrate induced cell cycle arrest in mammalian cells through an increase in p21^Waf1/Cip1, although another study showed that this arrest is related to pRB signaling. We isolated variants of HeLa cells adapted to growth in 5 mM butyrate. One of these variants, clone 5.1, constitutively expressed elevated levels of p21^Waf1/Cip1 when incubated in regular growth medium and in the presence of butyrate. Despite this elevated level of p21^Waf1/Cip1, the cells continue to proliferate, albeit at a slower rate than parental HeLa cells. Western blot analyses showed that other cell cycle regulatory proteins were not up-regulated to compensate for the elevated expression of p21^Waf1/Cip1. However, cyclin D1 was down-regulated by butyrate in HeLa cells but not in clone 5.1. We conclude that continued expression of cyclin D1 allowed clone 5.1 to grow in the presence of butyrate and elevated levels of p21^Waf1/Cip1.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Sodium butyrate added to cultured cells lead to growth arrest, cellular differentiation (1), or cell death (2-4). Butyrate is a natural by-product of a high fiber diet and has been reported to offer protection against colorectal cancer (5, 6). There is increasing interest in its potential as a chemotherapeutic agent against other forms of cancers (7-12). Cell cycle studies have indicated that the cytostatic effects of butyrate could be attributable to the up-regulation of p21^Waf1/Cip1 because disruption of this gene allowed colon cancer cells to continue dividing (13). However, using primary fibroblasts with disrupted p21^Waf1/Cip1, Vaziri et al. (14) showed that butyrate-induced cell cycle arrest could be independent of p21^Waf1/Cip1, instead involving cyclin D1 expression and pRB¹ signaling. They further suggested that there were at least two mechanisms of butyrate-induced arrest in cells involving the down-regulation of expression of cyclin D1 and the up-regulation of p21^Waf1/Cip1. Similar observations were also reported by Davis et al. (15).

During the mid-G₁ phase of the cell cycle, cyclin D1 activates cdk4 and cdk6 to phosphorylate pRB to release E2F, the transcription factor that induces genes required for cells to enter S phase (reviewed by Sherr (16)). P21^Waf1/Cip1 inhibits cell cycle progression by binding to and inhibiting cyclin-dependent kinase (cdk)/cyclin complexes (17-19), thereby maintaining the pRB in the dephosphorylated state. Up-regulation of p21^Waf1/Cip1 has been reported in some quiescent cells that were stimulated to re-enter cell cycle (20-22), and it was proposed that low levels of p21^Waf1/Cip1 may function to stabilize the cdk/cyclin complexes, whereas high levels were inhibitory (23, 24).

Although butyrate and trichostatin A are well known to inhibit cell division (1, 6, 25) and to induce apoptosis (2-4), variants can be isolated that proliferate in the presence of these inhibitors. Chalkley and Shires (34) isolated a hepatoma cell line that adapted to growth in 6 mM butyrate. These cells were established by continuous incubation in increasing concentrations of sodium butyrate. Human erythroleukemia cell lines resistant to the differentiation-inducing effects of butyrate were also isolated by Ohlsson-Wilhelm et al. (33). More recently, a mouse mammary cell line, TR303, was isolated for resistance to trichostatin A following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (26, 27). Other butyrate-resistant variants were isolated from HL60 promyelocytic leukemia cells (28, 29) and from HeLa S3 (30). In all these cases, cell cycle studies were not done, and the mechanism of their abilities to proliferate in butyrate-containing medium is unknown.

We have isolated a variant of HeLa cells that is resistant to high concentrations of butyrate. Cell cycle studies showed that this butyrate-resistant clone 5.1 constitutively expresses elevated levels of p21^Waf1/Cip1. In this study, we show that the expression of cyclin D1 allows this cell to continue to proliferate in the presence of 5 mM butyrate.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture and Isolation of Butyrate-resistant Clones-- HeLa cells and their butyrate-resistant clone 5.1 were routinely grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, penicillin, and streptomycin. For isolation of butyrate-resistant variants, a total of about 5 × 10⁷ HeLa cells was plated in 100-mm tissue culture dishes. After an overnight incubation period to allow the cells to attach, 1 M sodium butyrate (Sigma), dissolved in PBS, was added to the cultures to final concentrations of 5, 7.5, or 10 mM, and the cells were continuously maintained in their respective media with regular changes. The clones that grew were isolated and transferred to separate flasks and expanded in butyrate-free medium. These clones were again subjected to a second round of selection in supplemented Dulbecco's modified Eagle's medium containing 5 mM butyrate, and the resulting clones were isolated and expanded, and frozen stocks were made. One clone was put through two additional rounds of subcloning for purification, and this purified clone, clone 5.1, was used for cell cycle studies. Clone 5.1 was maintained in regular growth medium in the absence of butyrate. Routine bimonthly tests showed that the ability to grow in 5 mM butyrate was stable.

Cell Growth-- Cells were plated in 6-well plates at a density of 10-20 × 10⁵ cells/well and allowed to incubate overnight before the addition of butyrate. Cells from one set of wells were counted, and this was used as the starting cell number. After incubation in 1 or 5 mM butyrate for 1, 2, or 3 days, the cells were washed with PBS, harvested by trypsinization, and counted in a Coulter Particle Counter (Beckman Coulter). Samples were plated in duplicate, and the average of three counts was used for each well. The experiment was conducted three times.

Antibodies and Western Blot Analyses-- Polyclonal antibodies for cyclins D1, D2, D3, p16^INK4a, p21^Waf1/Cip1, p27^Kip1, and cdk6 were purchased from Santa Cruz Biotechnology Inc. Monoclonal antibodies to proliferating cell nuclear antigen (PCNA) and cyclin B were also purchased from Santa Cruz Biotechnology Inc. Polyclonal antibodies to cdk4 and p53 were purchased from Calbiochem. Monoclonal antibodies to cyclin E and pRB were purchased from PharMingen. Secondary goat -rabbit and goat -mouse antibodies conjugated with horseradish peroxidase were purchased from Pierce.

For experiments, cells were plated at ~80% confluence and treated with 0, 1, or 5 mM sodium butyrate overnight. The cells were then collected by scraping into the medium, pelleted by centrifugation, and washed once with cold PBS. Cell pellets were lysed in nuclear buffer (31), the nuclei were pelleted by centrifugation, and 1-5 µl of the supernatant was used for protein quantity determination using the Bio-Rad microassay with bovine serum albumin as the relative protein standard. 20 µg of total cellular protein was fractionated in a 12% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore). Immunostaining with primary and secondary antibodies was performed according to manufacturers protocols.

Immunoprecipitation and Kinase Assays-- Immunoprecipitation of cdk2 and in vitro kinase assays were performed according to the method described by Guo et al. (32) with some modifications. Briefly, cells were lysed in 400 µl of NB buffer containing Complete protease inhibitor (Roche Diagnostics) and 100 nM calyculin A as described above and centrifuged to remove insoluble material, including the nuclei. The supernatant was first precleared with 0.5 µl of preimmune serum and 75 µl of protein A-Sepharose 6MB suspension (Amersham Pharmacia Biotech). Specific antibody along with fresh protein A-Sepharose 6MB was added, and the mixture was rotated in the cold room for 2-3 h. The beads were washed six times with NB buffer before being used for kinase assays.

The protein A-Sepharose 6MB beads with immunoprecipitate were first equilibrated with 100 µl of H1 kinase buffer (50 mM Tris, pH 7.4, 2 mM MgCl₂, 1 mM dithiothreitol, 100 mM NaCl, and 0.05 mM ATP) for 10 min on ice. The buffer was removed and replaced with 50 µl of H1 kinase reaction buffer (H1 kinase buffer containing 0.25 M Na₃VO₄ and 100 nM calyculin A (Sigma), 0.1 µg/µl purified histone H1 (a gift from Dr. X. W. Guo (Helix Diagnostics)), and 0.05 µCi/µl [-³²P]ATP) and incubated at 30 °C for 10 min. The reaction mixture was then centrifuged to pellet the protein A-Sepharose beads, and the supernatant was transferred to a fresh tube. 45 µl of this supernatant was then added to nine volumes of cold acetone and placed at 20 °C for precipitation of histone H1. The samples were microcentrifuged for 5 min, and the supernatant was discarded. The histone H1 was air-dried, redissolved in SDS buffer, and electrophoresed in a 12% SDS-polyacrylamide gel. The protein was stained with Coomassie Blue, and the gel was dried and exposed to x-ray film for autoradiography to determine the levels of [³²P]phosphate incorporation.

To determine the relative amounts of proteins immunoprecipitated for kinase assays, the protein-Sepharose 6MB beads containing the immunoprecipitated proteins were resuspended in SDS buffer, fractionated with SDS-polyacrylamide gel electrophoresis, and immunostained for cdk2 as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolation of Butyrate-resistant Cells-- When HeLa cells are incubated with low concentrations (~1 mM) of sodium butyrate, they become arrested in their cell cycle. Increasing the concentration to 5 mM will induce apoptosis within 2 days. This effect of butyrate on cells is well documented (2-4). To isolate butyrate-resistant variants, HeLa cells were grown continuously in medium containing 5, 7.5, or 10 mM sodium butyrate with regular changes of medium. Most cells expired within a week of incubation. However, after 6 weeks of continuous incubation, three colonies appeared in plates with medium containing 5 mM butyrate, and one colony appeared in medium containing 7.5 mM butyrate. No survivors were isolated among cells incubated in 10 mM butyrate. Following another round of selection and further subcloning as described under "Materials and Methods," clone 5.1 was selected for further studies.

Cell Cycle Properties of Clone 5.1-- Clone 5.1 exhibited a reduced growth rate under normal growth conditions in regular growth medium with a doubling time of about 24 h, as compared with the 16-h doubling time for the parental HeLa cell. This is different from other butyrate-resistant cells that were reported to have growth rates similar to those of their parental cells (26, 28-30, 33, 34). In the presence of 1 mM butyrate, parental HeLa cells stopped dividing and remained in a state of quiescence (Fig. 1, left panel). Increasing the butyrate concentration to 5 mM resulted in complete cell killing within 3 days. In contrast, clone 5.1 continued to grow in butyrate, albeit at a reduced rate. In 1 mM butyrate, the cells took 3 days to double in density, and in 5 mM butyrate, the cell number increased by 50% in 3 days (Fig. 1, right panel). Clone 5.1 continued to grow in butyrate and formed visible colonies.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Butyrate effects on cell growth. HeLa (left panel) and clone 5.1 (right panel) cells were plated out in regular growth medium containing 0 (), 1 (), or 5 () mM butyrate. After incubating for up to 3 days, the cells were washed with PBS and harvested by trypsinization and then counted in a Coulter Counter.

The cell cycle arrest induced by butyrate was shown to result from the up-regulation of p21^Waf1/Cip1 (13), which then inhibited the activities of cyclin-dependent kinases (17-19, 35-37). However, another study by Vaziri et al. (14) showed that p21^Waf1/Cip1 up-regulation was not sufficient for cell cycle arrest and that cyclin D1 down-regulation also played a major role in this arrest. To examine the expression level of p21^Waf1/Cip1 in the presence of butyrate, immunoblot analyses of proteins from HeLa cells showed that p21^Waf1/Cip1 was low in cycling cells (Fig. 2, first lane) and was up-regulated with an overnight exposure to butyrate (Fig. 2, second and third lanes), leading to cell cycle arrest at G₁ and G₂ (Fig. 3). The percent of cells in S phase dropped from about 45% to less than 10% of total cells with incubation in 5 mM butyrate. In contrast, the butyrate-resistant clone 5.1 constitutively expressed elevated levels of p21^Waf1/Cip1 even when maintained in butyrate-free medium, and the inclusion of butyrate did not alter the expression of p21^Waf1/Cip1 (Fig. 2). Northern blot analyses showed that there was a corresponding increase in the mRNA encoding p21^Waf1/Cip1in clone 5.1 over the parental line (data not shown). Quantitation of transcript levels using TaqMan reverse transcription-polymerase chain reaction and analysis with an ABI 7700 sequence detection system (PE Applied Biosystems) showed that there was up to an 8-fold increase in clone 5.1 as compared with the parental line (data not shown). However, despite this elevation, cytofluorometric analyses showed that clone 5.1 retained a normal cell cycle profile with the percent of S phase cells remaining at around 40% (Fig. 3). In 5 mM butyrate, the cells continued to traverse through S phase but delayed progressing through G₂, potentially accounting for the reduced cell growth rates.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Expression of p21Waf1/Cip1. HeLa cells and the butyrate-resistant clone 5.1 were treated for 18 h with 0, 1, or 5 mM butyrate, and protein extracts were prepared as described under "Materials and Methods." Following electrophoresis in a 12% SDS-polyacrylamide gel and transfer onto a polyvinylidene difluoride membrane, immunoblot of p21Waf1/Cip1 was performed to determine the levels of expression.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Cell cycle arrest induced by butyrate. Cycling HeLa and clone 5.1 cells were incubated with butyrate as described in the legend for Fig. 1 and then harvested and fixed with 70% ethanol. The cells were then stained with propidium iodide and analyzed by flow cytometry.

In a study of human breast carcinoma by Balbín et al. (38), a mutation in p21^Waf1/Cip1 that converted an arginine to tryptophan at residue 94 resulted in its inability to inhibit cdks. In clone 5.1, cloning and sequencing of the cDNA encoding p21^Waf1/Cip1 did not reveal any sequence differences from that of the parental HeLa cell line (data not shown). The results suggest that during selection for butyrate resistance, clone 5.1 initially responded by up-regulating the expression of p21^Waf1/Cip1, and with a prolonged period of incubation, this variant overcame the inhibitory effects of this CKI and was able to enter and traverse S phase.

Expression of Cell Cycle Regulatory Proteins-- One possible way for clone 5.1 to overcome the inhibitory effects of p21^Waf1/Cip1 overexpression would be to concomitantly increase the expression of other cell cycle regulatory proteins. To determine whether there were compensatory increases in cell cycle proteins in clone 5.1, cell extracts were prepared following treatments with increasing concentrations of butyrate, and immunoblots were performed. As shown in Fig. 4, there was no increase in the expression of cyclin-dependent kinases. The expression of cdk2 and cdk6 were similar between the parental HeLa cells and clone 5.1 in the presence or absence of butyrate. A decrease in the expression of cdk4 was seen in both cell lines treated with butyrate. The expression levels of proliferating cell nuclear antigen and the cdk inhibitors p16^INK4a and p27^KIP were unaffected by butyrate in either cell. Western blot analyses also showed that the expression of pRB was lower in clone 5.1 when compared with HeLa cells, and these levels in either cell line were not affected by the presence of 5 mM butyrate.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Expression of cell cycle proteins. HeLa and clone 5.1 were treated with increasing concentrations of butyrate as described in the legend for Fig. 1, and whole cell extracts were prepared. Proteins were then separated and transferred onto a polyvinylidene difluoride membrane, and immunostained for expression of cell cycle regulatory proteins.

Immunoblot analyses showed a reduction in cyclin A levels in both cell lines in the presence of butyrate (Fig. 5). A similar decline in cyclin B level was seen in HeLa cells treated with butyrate but was unaffected by butyrate in clone 5.1. The expression of cyclin E was not affected by the presence of butyrate in either of the cell lines. However, cyclin D1 in HeLa cells was down-regulated by increasing concentrations of butyrate. In contrast, in clone 5.1, the expression of cyclin D1 was elevated and was only partially down-regulated by 5 mM butyrate. Measurement of transcript levels by reverse transcription-polymerase chain reaction and ethidium bromide staining and by the TaqMan reverse transcription-polymerase chain reaction system showed that the mRNA encoding cyclin D1 corresponded directly with the protein levels seen by immunoblot (data not shown). Quantitation showed that the presence of butyrate down-regulated cyclin D1 mRNA in HeLa cells by about 90%, whereas the cyclin D1 transcript level remained relatively constant in clone 5.1. The expression of cyclin D2 or D3 in either cell line was unaffected by the presence of butyrate. The results suggest that the elevated expression of cyclin D1 allowed clone 5.1 to overcome the inhibitory effects of butyrate.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   Expression of cyclins. Protein extracts from HeLa and clone 5.1 cells treated with butyrate were immunoblotted for cyclins as described in the legends for Figs. 1 and 3.

Histone H1 Kinase Activity of Cdk2-- P21^Waf1/Cip1 inhibits cell cycle progression by associating with and inactivating cdk/cyclin complexes. To examine these interactions in clone 5.1, p21^Waf1/Cip1 was immunoprecipitated from control cells and cells that were incubated overnight with 5 mM butyrate and then Western blotted for cdk2 and cyclin E. Fig. 6 shows that in HeLa cells treated with butyrate and in control or butyrate-treated clone 5.1, p33^cdk2 and cyclin E co-immunoprecipitated with p21^Waf1/Cip1. Enzymatic assays using these immunoprecipitated complexes extracted from either the HeLa or clone 5.1 revealed that the kinases did not have enzymatic activity using histone H1 as substrate (data not shown). This result shows that the p21^Waf1/Cip1 in clone 5.1 was functional in its ability to inactivate cdks.

View larger version (51K): [in this window] [in a new window]   Fig. 6.   Enzyme activity assays of kinase complexes associated with p21Waf1/Cip1. P21Waf1/Cip1 was immunoprecipitated, and the complexes were assayed for kinase activity with histone H1 as the substrate (panel A). Immunostaining of proteins that co-immunoprecipitated with p21Waf1/Cip1 (panel B) revealed the presence of cdk2 (panel C) and cyclin E (panel D).

Following depletion of p21^Waf1/Cip1from the cell extracts, we immunoprecipitated the remaining soluble p33^cdk2 from the cell extracts and assayed for kinase activities using histone H1 as the substrate. The results showed that p33^cdk2 in control HeLa cells had high histone H1 kinase activity (Fig. 7), but the p33^cdk2 kinase immunoprecipitated from the butyrate-treated HeLa cells was enzymatically inactive. This result corresponded with the cell cycle arrest seen in Fig. 3. It is likely that this inactive p33^cdk2 in the butyrate-treated cells was from the cells that were in early G₁, whereas the p33^cdk2 from cells that were in late G₁/S was inactivated by binding to p21^Waf1/Cip1. This is further suggested by the down-regulation of cyclin D1, which is expressed at mid-G₁. In clone 5.1, the p33^cdk2 that was not bound to p21^Waf1/Cip1 was enzymatically active both in the control and after butyrate treatment. The overexpression of cyclin D1 in these cells allows the activation of cdk2 to traverse the cell cycle. Furthermore, because the levels of cyclin A were reduced with butyrate treatment in all the cells, the major cdk2 activity in clone 5.1 was attributed to p33^cdk2/cyclin E (Fig. 4).

View larger version (42K): [in this window] [in a new window]   Fig. 7.   Kinase assay of cdk2 kinase. Following immunodepletion of p21Waf1/Cip1, cdk2 was immunoprecipitated from the cell extracts and assayed for histone H1 kinase activity (panel A). Immunostaining of the proteins that co-immunoprecipitated with cdk2 confirm the presence of p33cdk2 (panel B) and cyclin E (panel C).

During the cell cycle of normal cells, p33^cdk2 regulates entry into S phase by phosphorylating pRB, allowing release of transcription factor E2F, which stimulates the synthesis of proteins necessary for DNA replication (reviewed by Sherr (16)). To determine the in vivo activity of p33^cdk2, the phosphorylation state of pRB was examined in both cell lines in the absence or presence of butyrate. Western blot analyses showed that pRB is hyperphosphorylated in cycling HeLa cells, as evident from the reduced electrophoretic migration (Fig. 8). Upon up-regulation of p21^Waf1/Cip1 by butyrate, pRB was dephosphorylated. In clone 5.1, the pRB was in a hyperphosphorylated state, and this did not change with butyrate treatment, indicating that the cdks retained their activities despite the overexpression of p21^Waf1/Cip1. The results further confirm that the cdk2 in clone 5.1 remained enzymatically active in the presence of butyrate, thus allowing the cell cycle to continue.

View larger version (39K): [in this window] [in a new window]   Fig. 8.   Phosphorylation of pRB. Immunoblot of pRB isoforms in whole cell extracts from HeLa cells and clone 5.1 cells that were incubated overnight with and without butyrate are shown.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Recently there is an increase in interest in the potential of histone deacetylase inhibitors as chemotherapy agents; however, our understanding of their mechanisms of action is incomplete. This knowledge will be fundamental to the further development of such compounds. Butyrate is one such histone deacetylase inhibitor that has long been known to induce cell cycle arrest and differentiation in numerous cell lines. At sufficiently high concentrations, it can induce apoptosis. In this report, we show that HeLa cells, a prototypical transformed cell, can spontaneously develop resistance to butyrate. We isolated clone 5.1 from HeLa cells that do not undergo apoptosis in 5 mM butyrate but will continue to grow, although at a reduced rate. Immunoblots show that the cdk inhibitor p21^Waf1/Cip1 was constitutively expressed in this cell and that cyclin D1 was overexpressed, allowing the cells to grow in butyrate.

Abberant expressions of p21^Waf1/Cip1 and cyclin D1 were reported in a number of cancers. In IGROV1 ovarian cancer cells with abnormally high levels of p21^Waf1/Cip1, there was also the overexpression of other cell cycle proteins (41). In the MCF-7 cell line, the overexpression of cyclin D1 and D3 overcame the inhibitory effects of p21^Waf1/Cip1 overexpression. The overexpression of p21^Waf1/Cip1 was also reported in a number of human brain tumors, but the compensatory overexpression of other cell cycle proteins was seen in only a few samples (42). Cyclin D1 overexpression was also reported in a number of breast cancer tissues (43, 44).

In butyrate-treated HeLa cells, the p33^cdk2 was found to be enzymatically inactive even though it was not associated with p21^Waf1/Cip1 (Fig. 6, lane 2). It is most likely that this fraction of p33^cdk2 is from the subpopulation of cells that were arrested in early G₁ before they were activated by phosphorylation and association with cyclins in mid-G₁ (45, 46). This other checkpoint is most likely associated with the down-regulation of cyclin D1 by butyrate. This is consistent with the findings of Vaziri et al. (14), who used mouse 3T3 cells that were depleted of p21^Waf1/Cip to show that the butyrate was just as efficient in inducing G₁ arrest. The authors suggested that there was at least one other butyrate-induced checkpoint in early G₁ and that this additional checkpoint could involve cyclin D1. The role of cyclin D1 in regulating the G₁ phase of the cell cycle was also described by Pervin et al. (47), who showed that nitric oxide induced cell cycle arrest by down-regulating cyclin D1 expression.

In butyrate-treated clone 5.1, cyclin D1 expression was elevated and continued to be expressed in the presence of butyrate. The fraction of p33^cdk2 that was unbound to p21^Waf1/Cip1 remained enzymatically active and is most likely responsible for the cells traversing S phase and continuing with their cell cycle. This suggests that the expression of cyclin D1 allowed the cells to continue with their cell cycle. However, it is unlikely to involve the pRB signaling pathway as suggested by Vaziri et al. (14) because pRB in HeLa cells is inactivated by binding to the E7 protein of human papillomavirus (39, 40). Recent reports have indicated that cyclin D1 has other functions in the cell that do not involve pRB phosphorylation. Hirai and Sherr (48) showed that D-type cyclins could directly bind to DMP1, a myb-like transcription factor. In breast epithelial cells, cyclin D1 could activate the estrogen receptor to induce transcription of steroid-responsive genes (49, 50), suppress the initiation of skeletal muscle differentiation independent of pRB phosphorylation (51), and bind to the STAT3 transcription factor to down-regulate its activity (52). In Drosophila, cyclin D regulates cell growth independent of the pRB homolog, RBF (53). What role these factors may play in regulating G₁ in clone 5.1 is currently under investigation.

Butyrate inhibits histone deacetylases (HDACs), resulting in the hyperacetylation of histones (54, 55). Similar effects on histone hyperacetylation and cell cycle arrest were also seen when cells were treated with trichostatin A, a specific inhibitor of HDAC (26, 27). Previous studies on butyrate-resistant cells focused mainly on the activities of the histone deacetylases (27-30, 33, 34), and in each case, the HDACs were resistant to the presence of these inhibitors. With the HeLa cells described in this study, exposure to butyrate or trichostatin A caused their histones to be hyperacetylated to their tri- and tetra-acetylated states, indicating the high sensitivity of their HDACs to these inhibitors. In contrast, incubation of clone 5.1 with butyrate or trichostatin A did not lead to the similar hyperacetylation of histones. Although some H4 histones were tri- and tetra-acetylated, there were about equal amounts of unacetylated and monoacetylated histones, indicating that the HDACs remained active in these cells.²

Preliminary characterization of another butyrate-resistant clone, Clone 7.5, which was isolated by continuous incubation in 7.5 mM butyrate, showed that it also has up-regulated p21^Waf1/Cip1, did not overexpress other cell cycle proteins, and has p33^cdk2 kinase that remained enzymatically active in the presence of butyrate. However, Clone 7.5 showed some distinctions from clone 5.1, including an even much slower cell division rate (doubling time of about 36 h) and a much lower expression of cyclin B. Further characterizations of this clone are under way.

	ACKNOWLEDGEMENTS

We thank Alison Graver of the Paleo-DNA Laboratory in Lakehead University for sequencing the cDNAs of p21^Waf1/Cip1 and cdk2. We also thank the Northern Cancer Research Foundation for their support.

	FOOTNOTES

^* This work was supported by a grant from the Medical Research Council of Canada (to J. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be sent: Northwestern Ontario Regional Cancer Centre, 290 Munro St., Thunder Bay, Ontario P7A 7T1 Canada; Tel.: 807-343-1542; Fax: 807-343-1549; E-mail: john.th'ng@cancercare.on.ca.

Published, JBC Papers in Press, July 26, 2001, DOI 10.1074/jbc.M104735200

² A. Derjuga, C. Richard, M. Crosato, P. S. Wright, and J. P. H. Th'ng, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: pRB, retinoblastoma protein; cdk, cyclin-dependent kinase; PBS, phosphate-buffered saline; HDAC, histone deacetylase.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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