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[Cancer Research 62, 67-74, January 1, 2002]
© 2002 American Association for Cancer Research

Biochemistry and Biophysics

Deregulation of Polyamine Biosynthesis Alters Intrinsic Histone Acetyltransferase and Deacetylase Activities in Murine Skin and Tumors1

Cheryl A. Hobbs, Barry A. Paul and Susan K. Gilmour2

Lankenau Institute for Medical Research, Wynnewood, Pennsylvania 19096


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The essential requirement for polyamines for normal cell growth and differentiation may be partly attributed to their influence on gene expression, a process regulated by the acetylation state of nucleosomal histones. We used transgenic mice to examine the effects of constitutive expression of ornithine decarboxylase (ODC), a key rate-limiting enzyme in polyamine biosynthesis, on histone acetylation in epithelial cells in skin. As compared with the skin of normal littermate mice, both intrinsic histone acetyltransferase (HAT) and deacetylase activities are elevated in ODC transgenic skin. Skin tumors that form spontaneously in ODC/Ras double transgenic mice exhibit exceptionally high HAT activity having a distinct specificity for Lys-12 in the tail domain of histone H4, which may have implications for gene transcription. However, acetylation of histones by HAT enzymes was impeded in cultured ODC transgenic keratinocytes, and there were only modest changes in levels of acetylated histones in the skin of ODC transgenic mice. Treatment with the ODC enzyme inhibitor {alpha}-difluoromethylornithine, which results in regression of ODC/Ras tumors, reverses the effects on HAT and deacetylase enzyme function, implicating polyamine biosynthesis in the regulation of histone acetylation. Polyamines do not directly stimulate the enzymatic activity of either p300 or p300/CREB-binding protein (CBP)-associated factor, members of two distinct classes of HAT enzymes, implying that the elevated CBP/p300-associated HAT activity detected in ODC transgenic skin is attributable to indirect influence of polyamines. These results suggest that multiple mechanisms exist by which endogenous polyamines influence chromatin in mammals. Furthermore, they suggest that the elevated polyamine levels inherent in many solid tumors alter chromatin structure, likely affecting gene expression and promoting the neoplastic process.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent discoveries have established that the acetylation status of nucleosomal histones is fundamental to the transcriptional competency of chromatin (1, 2, 3, 4, 5) . The acetylation state of histones is determined by the equilibrium established between two general classes of enzymes, HATs3 and HDACs. The acetylation of lysine residues in the NH2-terminal tail domain of core histones destabilizes nucleosomes, allowing greater access of DNA to transcription factors (6) . Conversely, deacetylation of histones typically stabilizes a transcriptionally repressed state. The HAT and HDAC enzymes are directed to gene promoters through interactions with large multiprotein complexes that are recruited by DNA-bound regulatory factors (7 , 8) . Functional specificity is governed by the specific combination of components that comprise the multisubunit HAT and HDAC complexes (9) . Little is known about how the HAT and HDAC enzymes themselves are regulated to generate the dynamic changes in the acetylation patterns of histones necessary for appropriate transcriptional responses to various biological conditions. Exchange of HAT and HDAC complex components, leading to a switch in transcriptional activation state, can be induced through mitotic stimulation or the binding of ligands to nuclear receptors (10, 11, 12) . Changes in the phosphorylation status and acetylation of HAT proteins themselves may affect enzymatic activity (13, 14, 15, 16) . However, to date, the short-chain fatty acid butyrate, a by-product of fermentation of dietary wheat fiber in the colon, is the only known nonprotein modulator of histone acetylation synthesized naturally in mammals.

Accumulating evidence implicates the inappropriate acetylation or deacetylation of histones in cell transformation. A variety of malignancies have now been identified, particularly acute leukemias, which are associated with defects in either HAT or HDAC genes, or in the recruitment of HAT or HDAC enzymes to gene regulatory regions (17, 18, 19, 20, 21, 22) . Correlating increases in histone acetylation and cellular levels of polyamines have been linked with proliferation (23) . This suggests that the small cationic polyamines may promote chromatin remodeling and altered expression of growth-related genes. In fact, transcription of several genes associated with cell proliferation, including the proto-oncogenes c-myc, c-jun and c-fos, appears to be modulated by polyamines (24 , 25) . We have found that the transcriptional activity of a variety of mammalian and viral gene promoters is enhanced in epidermal cells that have been retrovirally engineered to constitutively express ODC (26) , the enzyme responsible for catalyzing the first step in polyamine biosynthesis.

Up-regulation of ODC expression and corresponding elevated levels of polyamines are characteristic of many solid tumors. Although aberrant expression of ODC is not generally sufficient to cause transformation of normal fibroblasts or keratinocytes, it does cooperate with genetic mutations in epidermal cells to promote tumor formation and invasiveness (27 , 28) . Accordingly, constitutive expression of ODC directed to hair follicles of transgenic mice (29) does not give rise to tumors, but is sufficient to drive spontaneous formation of papillomas in the skin of mice that also bear a v-Ha-ras transgene (30) . Skin tumors begin to develop in these ODC/Ras double transgenic mice at 5–8 weeks of age and exhibit a high frequency of conversion to malignant carcinomas. Treatment with DFMO, a specific inhibitor of ODC enzymatic activity, causes these tumors to quickly regress, demonstrating that polyamine synthesis is required for tumor maintenance.

The correlation of elevated intracellular polyamine levels with altered histone acetylation and gene transcription suggests that polyamines may affect one or more of the enzymes responsible for acetylating or deacetylating histones. In fact, the activity of HATs and HDACs has been shown to be modulated by the addition of spermidine and spermine in cell-free assays (31, 32, 33) . However, experiments relying on exogenous addition of polyamines to some subcellular fraction may not adequately reflect the situation in vivo (26 , 34) , probably reflecting the compartmentalization of polyamines into distinct subcellular pools, which vary according to the biological status of the cell. We recently reported that elevated intracellular levels of polyamines directly or indirectly promote the activity of HAT enzymes and hyperacetylation of histones in NIH3T3 and epidermal cell lines retrovirally engineered to overexpress ODC (35) . We have extended our investigation to a more relevant physiological system in which cells undergo proliferation and differentiation within their natural tissue context. Using the K6/ODC (29) and ODC/Ras (30) transgenic mouse models, we find enhanced intrinsic activities of HAT and HDAC enzymes and altered HAT function in ODC-overexpressing skin and isolated keratinocytes. Treatment with the ODC enzyme inhibitor DFMO reverses these effects, specifically implicating polyamine biosynthesis in the direct or indirect regulation of these enzymes. Significantly, the skin tumors that develop spontaneously in ODC/Ras bigenic mice have exceptionally high HAT activity. Although p300/CBP-associated HAT activity is elevated in ODC transgenic skin, it does not appear to contribute to this elevated tumor HAT activity. Interestingly, the HAT activity in ODC transgenic skin and tumors exhibits a distinct substrate preference for Lys-12 in the tail domain of histone H4, which may be indicative of transcription-associated hyperacetylation of histones (36) , or formation of heterochromatin (37 , 38) and gene silencing (39) . Thus, elevated ODC activity likely has significant, possibly even opposing, ramifications for gene expression. These results provide the first evidence of multiple polyamine-mediated effects on enzymes that regulate the acetylation status of histones in higher eukaryotes, and suggest a general mechanism by which aberrant polyamine biosynthesis contributes to cell transformation.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic Animals.
K6/ODC mice on FVB or C57BL/65 genetic backgrounds were bred with TG.AC transgenic mice on a FVB background to produce ODC/Ras double transgenic progeny (30) . Tumors begin to develop spontaneously on ODC/Ras mice at approximately 5 weeks of age. Littermate mice were used for individual experiments. As such, typically n = 1–3 mice for each treatment category. The results provided are representative of multiple experiments performed for each study.

Culturing of Primary Keratinocytes.
K6/ODC transgenic pups (2–3 days old) were distinguished from their normal littermates by PCR genotyping. Primary keratinocytes were derived from the skin of these newborn pups by a procedure involving trypsin flotation (40 , 41) . The isolated keratinocytes were then cultured in 60-mm dishes in epidermal medium containing 8% chelexed fetal bovine serum, 5 ng/ml epidermal growth factor, and 0.05 mM calcium. The K6/ODC keratinocytes were propagated for approximately 5 days in the presence of 25 µM {alpha}-DFMO (Ilex Oncology) to titrate down ODC activity, thereby improving their viability. DFMO was then withdrawn for several days, producing a rapid rise in ODC enzymatic activity. To induce differentiation, keratinocytes were incubated for 24 h in medium containing 0.125 mM calcium.

Preparation of Skin and Tumor Homogenates.
Skins excised from ODC transgenic and normal littermate mice were immersed in 55°C H2O for 20 s to facilitate separation of the epidermis from the dermis. While kept cold, the epidermis was scraped from the dermis, and the dermis was cut into small pieces. The tissue was then frozen in liquid nitrogen, pulverized, and stored at -80°C. Spontaneous tumors from ODC/Ras double transgenic mice were frozen in liquid nitrogen and stored at -80°C. Frozen tumor tissue was pulverized just prior to use. Ground tissue was homogenized in RIPA buffer [50 mM Tris-HCl (pH 7.5), 1% NP40, 0.25% sodium deoxycholate, 0.25% SDS, 150 mM NaCl, and 1 mM EGTA] containing 2 µg/ml each aprotinin, leupeptin, and pepstatin, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 0.4 mM Pefabloc (Boehringer Mannheim), and 10 mM sodium butyrate by passing through a syringe needle after a 60-min incubation on ice. Ground tumor and skin tissues used in HAT and HDAC assays were homogenized in Tween 20 lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 2.5 mM EDTA, 0.01% Tween 20, 1 mM DTT, 10 mM ß-glycerophosphate] containing the protease and phosphatase inhibitors. Debris was removed by centrifugation, and the tissue extracts were stored at -80°C. Protein in tissue extracts was separated by SDS-PAGE, transferred to nitrocellulose and subjected to immunoblot analyses using antibodies directed against p300/CBP (PharMingen), HDAC-1 (Upstate Biotechnology), and ß-actin (Sigma Chemical Co.).

Analysis of Histones.
To radiolabel acetylated histones, cells were incubated for 1 h in conditioned medium containing 1 mCi/ml [3H]acetic acid (4.1 Ci/mmol; DuPont/NEN) and 10 mM sodium butyrate. Cells were then immediately placed on ice, washed three times with cold PBS containing 10 mM sodium butyrate, and frozen at -80°C. Cells were harvested by scraping in ice-cold lysis buffer (10 mM Tris-HCl pH 6.5, 50 mM sodium bisulfite, 1% Triton, 10 mM MgCl2, 8.6% sucrose) containing 10 mM sodium butyrate and 2 µg/ml each of aprotinin, leupeptin, and pepstatin, 1 mM sodium fluoride, 1 mM sodium orthovanadate, and 0.4 mM Pefabloc (Boehringer Mannheim). To isolate histones from skin, ground tissue was homogenized in the lysis buffer using a polytron. Nuclei were prepared by 20 strokes in a Dounce glass homogenizer using a tight-fitting pestle and then were washed three times in cold lysis buffer and one time with a cold solution containing 10 mM Tris-HCl (pH 7.4), 13 mM EDTA, 10 mM sodium butyrate, and protease and phosphatase inhibitors. The nuclei were resuspended in ice-cold H2O and briefly vortexed, and acid-insoluble material was precipitated by addition of H2SO4 to 0.4 N and incubation on ice for at least 1 h. After centrifugation, the acid-soluble protein in the supernatant was precipitated with 10 volumes of acetone and allowed to aggregate overnight at -20°C. Precipitated protein was collected by centrifugation, air dried, resuspended in H2O, and stored at -80°C.

Histones were separated by electrophoresis on 15% SDS-PAGE gels. For fluorography, gels were stained with Gelcode Blue reagent (Pierce) to verify uniform loading of histones, treated with Entensify (DuPont/NEN), dried on a gel dryer, and exposed to film using a BioMax TranScreen-LE intensifying screen (Kodak) at -80°C. For immunodetection of histones, protein was transferred to nitrocellulose filters, briefly stained with Ponceau S (Sigma Chemical Co.) to verify transfer of equivalent amounts of histone protein, and histones were detected using polyclonal antibodies against acetylated or total histones H3 or H4 (Upstate Biotechnology and Santa Cruz Biotechnology). Bound antibody was visualized using enhanced chemiluminescence (DuPont/NEN).

HAT and HDAC Assays.
To assay for HAT activity, tissue homogenates were incubated at 30°C for 30 min in 50 µl of IPH buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.05% NP40] containing 25 µg of core histones (Sigma Chemical Co.) or 1 µg of a synthetic H4 peptide (Upstate Biotechnology), 0.3 µCi [3H]acetyl-CoA (4.4 Ci/mmol; DuPont/NEN), 200 nM trichostatin A, and 0.4 mM Pefabloc. Each reaction mixture was spotted on a piece of Whatman P-81 phosphocellulose paper, which was then washed several times in 0.05 M NaHPO4-Na2PO4 (pH 9.2) at 37°C, washed once in acetone and once in methanol/chloroform (1:2), and air-dried prior to scintillation counting. For visual inspection of in vitro-labeled histone substrate, [14C]acetyl-CoA (4–6 Ci/mmol; DuPont/NEN) was substituted for the [3H]acetyl-CoA, and a portion of the reaction mixture was separated by SDS-PAGE and subjected to fluorography as described above. To assess p300/CBP-associated HAT activity, tissue extracts were incubated with a monoclonal antibody against p300/CBP (PharMingen) in IPH buffer at 4°C, and bound protein was collected by incubation with Protein G Plus/Protein A-agarose. After multiple washes with IPH buffer, the beads were resuspended in the HAT assay reaction mix, incubated at 30°C for 30 min, transferred to P-81 filters, and processed as described above. For analysis of direct effects of polyamines on p300 and PCAF, 200 ng of recombinant glutathione S-transferase (GST)-p300 fusion protein (aa1195–1707; Upstate Biotechnology) or 5 ng of recombinant GST-PCAF (aa352–832; Upstate Biotechnology) were incubated at 30°C for 30 min in HAT assay reaction mix containing 2 µg of core histones and various concentrations of spermidine, spermine, and putrescine (Sigma Chemical Co.). The reactions were analyzed by scintillation counting as described above. For in vitro measurement of HDAC activity, tissue homogenates were incubated at room temperature overnight in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% glycerol and 5 mM Pefabloc in the presence of a synthetic [3H]acetylated histone H4 peptide (prepared using the HDAC Deacetylase Assay kit; Upstate Biotechnology). Released 3H was extracted in ethyl acetate and measured by scintillation counting. The values reported are the averages of duplicate HAT and HDAC reactions after subtraction of the background level of counts in control reactions performed in the absence of tissue homogenate.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Elevated HAT Activity in ODC Transgenic Mouse Skin and Tumors.
We used our transgenic mouse models to examine the consequences of ODC overexpression and the resulting high concentrations of polyamines on the activity of HAT enzymes in skin. Using in vitro enzyme assays, we evaluated the overall HAT activity present in homogenates of skin from K6/ODC transgenic mice and their normal littermates. Fluorographic analyses of radiolabeled products of HAT reactions revealed that extracts of skin tissue from K6/ODC transgenic mice had substantially higher HAT activity toward core histones provided as substrate as compared with extracts of normal littermate skin (Fig. 1A)Citation . Although all four core histones were acetylated to some degree, histone H4 was acetylated to a much greater extent than the other histones. As measured by filter-binding assays, the enhancement in intrinsic HAT activity in ODC transgenic skin is typically ~3-fold but has been measured as high as 18-fold (data not shown).



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  		Fig. 1. Elevated levels of intracellular polyamines enhance intrinsic HAT activity in skin and tumors. A, HAT activity was compared between homogenates of whole skin from K6/ODC and normal littermate mice, and ODC/Ras and normal littermate epidermis and dermis. Core histones provided as substrate were labeled in HAT reactions, separated by SDS-PAGE, and examined by fluorography. Products of HAT reactions using ODC/Ras tumor homogenates are also shown. B, HAT activity in homogenates of K6/ODC and normal littermate epidermis and dermis and ODC/Ras spontaneous tumors was measured in a filter-binding assay using a synthetic peptide substrate corresponding to the NH2 terminus of histone H4. C, HAT activity in homogenates of skin from normal mice, tumors and adjacent nontumorigenic skin from ODC/Ras mice, and skin and regressing tumors from ODC/Ras mice treated with DFMO, was measured using the H4 peptide substrate. The tumor data reflect the averages of independent HAT assays of several tumors from both nontreated and DFMO-treated mice. D, p300/CBP was immunoprecipitated from homogenates of whole skin from K6/ODC and normal littermate mice, and the associated HAT activity was assayed using a synthetic histone H4 peptide as the substrate. HAT activity was normalized for the amount of p300/CBP protein present in the original homogenates as measured by immunoblot analysis. E, HAT enzymatic activity of purified recombinant p300 and PCAF was measured in the presence of various concentrations of spermidine and putrescine using core histones as substrate. Results of control reactions for nonenzymatic acetylation of histones and polyamines are also shown. NA, not assayed.

 
Mice generated from breeding K6/ODC transgenic mice with mice homozygous for a v-Ha-ras allele (TG.AC) spontaneously form skin tumors without any prior treatment with a chemical initiating or promoting agent (30) . These spontaneously forming tumors quickly regress on administration of DFMO, a specific inhibitor of ODC enzymatic activity, in the drinking water. Activated ras continues to be expressed in these regressing tumors (42) , arguing that polyamine biosynthesis is a critical event required for tumor maintenance. We compared HAT activity in extracts of epidermis and dermis of ODC/Ras double transgenic mice with that of their normal littermates. As observed for K6/ODC skin, fluorographic analysis of HAT reaction products demonstrated increased labeling of the core histone substrates, particularly histone H4, by the HAT activity present in ODC/Ras skin (Fig. 1A)Citation . Increased HAT activity was observed in both the epidermis and dermis, as well as in intact skin, of both K6/ODC and ODC/Ras mice (Fig. 1, A–CCitation , and data not shown). Using either core histones or a histone H4 peptide as substrate in filter binding assays, dramatically higher levels of HAT activity were measured in spontaneous tumors from ODC/Ras transgenic mice relative to normal and K6/ODC skin, as well as nontumor-bearing skin from the same ODC/Ras mice (Fig. 1, B–C)Citation . Thus, although overexpression of ODC appears to enhance in vitro acetylation of core histones by HAT enzymes, there exists an additional enhancement effect on acetylation of histone H4 that is specific to the ODC/Ras tumors.

As a consequence of DFMO treatment, HAT activity in ODC/Ras skin generally decreased, and the aberrantly high HAT activity in the tumors was reduced to a level comparable with that detected in nontreated skin (Fig. 1C)Citation . This depression of intrinsic HAT activity was seen after only 3 days of DFMO treatment. Because the regressing tumors continue to express v-Ha-ras, the decreased HAT activity observed in these tumors must be specifically attributable to the decreased levels of polyamines resulting from DFMO treatment. These studies clearly demonstrate that intrinsic HAT activity in both the skin and the tumors of ODC transgenic mice is dependent on intracellular polyamine levels.

The CBP and p300 transcriptional coactivator proteins play a pivotal role in integrating multiple signaling pathways (43 , 44) . In some instances, their intrinsic HAT activities are required for gene activation (9) , presumably for mediating alterations in the local chromatin structure. Because it is conceivable that polyamine-mediated modulation of CBP/p300 function could account for many of the diverse effects exerted by polyamines on cell growth and differentiation, we examined the effect of overexpression of ODC in skin on CBP/p300 HAT activity. Typically, there was an increase in HAT activity associated with CBP/p300 immunoprecipitated from extracts of K6/ODC transgenic skin tissue as compared with normal littermate skin (Fig. 1D)Citation . However, relative to K6/ODC skin or nontumor-bearing ODC/Ras skin, there was no apparent increase in the ability of CBP/p300 protein complexes in several examined tumors to acetylate the histone H4 substrate (data not shown). Moreover, in contrast to the situation for skin, the CBP/p300-associated HAT activity in the few tumors from treated animals that we examined did not appear responsive to DFMO treatment (data not shown). Thus, although CBP/p300 HAT function is increased in ODC-overexpressing tissue, the exceptionally high histone H4-specific HAT activity characteristic of tumors in ODC/Ras transgenic mice is not primarily contributed by CBP/p300 or associated HAT enzymes, but by some other independent HAT activity.

Because p300/CBP-associated HAT activity is elevated in K6/ODC transgenic mouse skin, we assessed the ability of polyamines to directly modulate the acetyltransferase activity of purified recombinant p300. The addition of spermidine and putrescine resulted in a dose-dependent inhibition of p300 HAT activity at concentrations that are high relative to estimated concentrations of these polyamines in mammalian cells (Fig. 1E)Citation . The coactivator/HAT enzyme PCAF is known to associate with p300/CBP complexes in some circumstances (45) . Thus, enhanced activity of PCAF could contribute to the elevated HAT activity associated with p300/CBP immunoprecipitated from K6/ODC skin extracts. Therefore, we also examined the effects of the individual polyamines on PCAF acetyltransferase activity (Fig. 1E)Citation . Spermidine and putrescine exerted some inhibitory effect on PCAF, but only at a very high polyamine concentration. Spermine had no direct effect on either p300 or PCAF HAT activity even at very high concentrations (data not shown). Importantly, these experiments demonstrate that polyamines do not directly stimulate the catalytic domains of representatives of two distinct classes of HAT enzymes.

Preferential Acetylation of H4 Lys-12 by HAT Activity in ODC Skin and Tumors.
We used a panel of histone H4 peptide substrates that were acetylated at various lysine residues (Fig. 2A)Citation to examine the acetylation specificity for the HAT activity present in ODC/Ras tumors. As compared with reactions using a control peptide that can be acetylated at all four lysine residues, the very low HAT activity measured in reactions using a peptide containing an acetylated Lys-12 residue indicates that Lys-12 is a preferred site of acetylation in histone H4 by the HAT activity present in the tumor extracts (Fig. 2B)Citation . In contrast, the peptide preacetylated at Lys-5 was acetylated almost to the same extent as the control peptide, indicating that the tumor HAT activity does not appreciably acetylate Lys-5 in histone H4. To confirm that there was no inherent bias against acetylation of the Ac-Lys-12 peptide in this assay system, an identical assay was performed using purified PCAF. Consistent with other reports (46 , 47) , PCAF exhibited a marked preference for Lys-8 of histone H4, but showed little decrease in acetylation of the Ac-Lys-12 peptide relative to the control peptide (data not shown). The distinct preference for Lys-12 is specifically related to the high levels of ODC, rather than to expression of activated Ras because a similar pattern of acetylation was exhibited by HAT enzymes in K6/ODC skin homogenates (Fig. 2C)Citation .



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  		Fig. 2. Lysine-12 in histone H4 is a preferred site of acetylation by the HAT activity in K6/ODC skin and ODC/Ras tumors. Tissue homogenates were assayed for HAT activity using various histone H4 peptides as substrate. A, the peptides used were either nonacetylated, acetylated at all four lysine residues, or acetylated at one of the four lysine residues as indicated. Decreased incorporation of 3H into an acetylated peptide relative to that of the nonacetylated peptide substrate indicates that the specific lysine is normally acetylated by the HAT activity present in the tissue extract. The tetra-acetylated histone H4 peptide serves as a negative control. B, homogenates from tumors were pooled (3 tumors/pool) and HAT activity assayed for lysine specificity. C, lysine specificities of HAT activities in homogenates of K6/ODC and normal littermate skin were compared.

 
Increased HDAC Activity in ODC Transgenic Skin.
We also examined the effect of constitutive overexpression of ODC on deacetylase activity in the skin and tumors of transgenic mice. HDAC activity was generally found to be increased in the skin of K6/ODC mice as compared with normal littermate controls (Fig. 3A)Citation . HDAC-1 has been identified as a component of a variety of multicomponent protein complexes that associate with chromatin (48, 49, 50, 51) . Because of its apparent predominance in the cell as compared with other HDACs, we compared the level of HDAC-1 protein in ODC transgenic and normal littermate skin. There was no obvious change in the level of HDAC-1 in K6/ODC transgenic tissue as assessed by immunoblot analyses (Fig. 3B)Citation . However, we cannot rule out that the elevated deacetylase activity in ODC skin reflects changes in the level of one or more of the many other HDAC enzymes identified to date. Interestingly, in contrast to HAT activity, tumor extracts exhibited no greater deacetylase activity toward the histone H4 substrate as compared with extracts of normal and ODC/Ras skin (Fig. 3C)Citation . In fact, HDAC activity in tumors was typically lower than that present in adjacent nontumor-bearing skin of ODC/Ras double transgenic mice (Fig. 3D)Citation . Treatment with DFMO resulted in decreased intrinsic HDAC activity in the skin of ODC/Ras mice (Fig. 3D)Citation . Thus, like HAT activity, HDAC activity is also susceptible to regulation by polyamine biosynthesis. However, HDAC activity in tumors did not appear to be responsive to DFMO treatment (Fig. 3D)Citation .



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  		Fig. 3. Intrinsic HDAC activity in skin is regulated by polyamine synthesis. The amount of HDAC activity present in tissue homogenates was measured as the release of 3H from a radiolabeled histone H4 peptide. A, HDAC activity was measured in K6/ODC and normal littermate skin. B, the amount of HDAC-1 in total protein extracts of K6/ODC and normal littermate mouse epidermis was examined by immunoblot analysis. The filter was reprobed for ß-actin to verify equivalent amounts of protein in each lane. C, HDAC activity in homogenates of ODC/Ras tumors was compared with that in adjacent nontumor-bearing skin and skin of normal mice. D, ODC/Ras mice were treated with DFMO and HDAC activity was compared between nontumor-bearing skin and tumors of treated and untreated mice.

 
Altered Acetylation of Histones in Cultured Keratinocytes and Skin of K6/ODC Transgenic Mice.
Because the in vitro assays detected elevated intrinsic HAT and HDAC activities in the skin of ODC-overexpressing transgenic mice, we assessed the effects of ODC overexpression on acetylation of histones in vivo. Primary keratinocytes cultured from K6/ODC transgenic and normal littermate skin were incubated with radiolabeled acetate in the presence of sodium butyrate, an inhibitor of deacetylase activity. Histones were then isolated and examined by fluorography and by immunoblot analysis (Fig. 4A)Citation . Interestingly, overall acetylation of histones in K6/ODC keratinocytes was found to be reduced as compared with control keratinocytes. Raising the calcium concentration to induce differentiation had no effect on the reduced level of acetylation mediated by constitutive expression of ODC. Treatment of the K6/ODC keratinocytes with DFMO restored acetylation of histones to normal levels, verifying its dependence on polyamine levels. Because deacetylase activity was blocked with sodium butyrate in these labeling experiments, these results suggest that polyamines directly or indirectly inhibit the ability of HAT enzymes to acetylate histones in normal diploid keratinocytes. A reciprocal analysis of polyamine effects specifically on HDAC function in proliferating and differentiating keratinocytes must await the identification of cell-permeable inhibitors of HAT enzymatic activity.



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  		Fig. 4. Overexpression of ODC alters acetylation of histones in keratinocytes and skin. A, primary keratinocytes cultured from K6/ODC and normal littermate mice were labeled with [3H]acetate for 1 h in the presence of 10 mM sodium butyrate. Isolated histones were separated by SDS-PAGE and examined by fluorography. Equal loading of histones was verified by staining with a Coomassie reagent. Histones were also subjected to immunoblot analysis using an antibody directed against acetylated histone H4. In a separate experiment, histones were isolated from keratinocytes induced to differentiate by treatment for 24 h in 0.125 mM calcium. Histones from parallel dishes of K6/ODC keratinocytes treated with DFMO to maintain ODC activity at normal levels are also shown. Additional experiments have determined that there is no noticeable difference in the overall level of acetylated histones in normal keratinocytes induced to differentiate as compared with those actively proliferating (data not shown). B, isolated histones from epidermis, dermis and total skin of K6/ODC and normal littermate mice were examined by immunoblot analyses using various antibodies directed against acetylated histones H3 and H4. Filters were reprobed with antibodies against histone H3 and H4 for normalization of the total amounts of histone protein loaded in each lane of the gels. Purified histones corresponding to some of the filter sets are shown stained with Coomassie reagent.

 
Unlike the in vitro HAT assays, these keratinocyte labeling studies measured polyamine-mediated effects on HAT function on the chromatin residing within the natural context of the living cell. In this case, the chromatin itself is susceptible to polyamine-mediated effects, in addition to the effects exerted directly or indirectly on HAT enzymes. Moreover, any effects of polyamines on the targeting of HAT complexes to histones might be reflected in the results of the labeling experiments but probably would not be detected in the artificial situation inherent of the cell-free assays that are conducted in the absence of endogenous nucleosomes or DNA. The lack of chromatin in these latter in vitro assays ensures that only changes in the intrinsic enzymatic activity of a sample are reflected.

To assess the effects of ODC overexpression on the overall level of acetylated histones in skin, histones were isolated from normal and K6/ODC transgenic skin and examined by immunoblot analyses using antibodies specific for acetylated histones. Although noticeable differences in acetylation were not always detected, consistent differences were observed in several independent histone preparations. In those cases, fewer acetylated histones H3 and H4 were detected in K6/ODC epidermal tissue, whereas acetylation of histones H3 and H4 was increased in K6/ODC dermis and total skin as compared with histones in normal littermate skin (Fig. 4BCitation and data not shown). Changes in the extent of acetylation of Lys-12 in histone H4 paralleled those observed for hyperacetylated histone H4.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To date, only a few inhibitors of HATs and HDACs have been identified, of which, only the short chain fatty acid butyrate, a by-product of wheat fiber fermentation in the colon, is normally synthesized in mammals. The ubiquitous polyamines, which serve a variety of cellular functions involved in regulating growth and differentiation, represent the first class of nonprotein molecules that naturally modulate histone acetylation in mammals. Previous studies of the effects of polyamines on HAT and HDAC function have been limited by their reliance on exogenous addition of individual polyamines to cell-free extracts (31, 32, 33) or to polyamine-depletion (52) , which eventually leads to growth arrest. The use of mice genetically engineered to overexpress ODC provides a means of assessing the involvement of polyamines in modulating HAT and HDAC function within the natural context of mammalian tissue. Moreover, the ODC/Ras double transgenic mouse provides a unique opportunity for correlating processes regulating histone acetylation with real-time tumor formation and regression.

Dysfunction of proteins involved in chromatin remodeling has been implicated in human disease and malignancy (17, 18, 19, 20, 21, 22) . Our studies point to a novel mechanism by which chromatin structure may become altered in mammals. Deregulation of polyamine biosynthesis resulting in altered HAT and HDAC enzyme function likely affects gene transcription and may additionally perturb other cellular processes that are associated with specific patterns of acetylation of lysines in histones, including DNA replication and cell differentiation. In reality, altered acetylation of histones resulting from changes in intracellular polyamine levels could occur much more often than as a consequence of gene mutations or chromosomal rearrangements involving HAT or HDAC enzymes. Aberrant expression of ODC and other genes involved in regulating polyamine metabolism is frequently associated with tumor progression in humans (53 , 54) . Moreover, polyamines can both promote (35) and inhibit (Fig. 4)Citation the acetylation of histones in epidermal cells, a feat presumably accomplished through different mechanisms. This, along with the observation that polyamines modulate both HAT and HDAC classes of enzymes, imply the existence of multiple regulatory controls on histone acetylation that are susceptible to polyamine influence. Indeed, restoration of normal acetylation patterns may be one downstream mechanism by which DFMO exerts its chemopreventative (55) and chemotherapeutic (30 , 42) effects, and may provide insight into the mechanism of action of some other anticancer drugs currently in clinical trials, including some polyamine analogues.

Polyamines bind and induce structural changes in DNA and chromatin (52 , 54 , 56 , 57) . Our data demonstrate that polyamines additionally affect HAT and HDAC enzymatic activities that also influence chromatin architecture. Conceivably, polyamines may modulate enzymatic activity by directly binding to HAT and/or HDAC proteins. However, neither purified p300 nor PCAF was directly stimulated by the addition of individual polyamines in cell-free assays. Therefore, at least in the case of p300/CBP protein complexes, it does not seem likely that the enhanced HAT activity measured in K6/ODC skin is caused by direct interaction of polyamines with the enzyme. Indeed, the situation with p300 and PCAF, representatives of two of several distinct families of nuclear HAT enzymes, suggest that polyamines may indirectly modulate the intrinsic activity of HAT enzymes in ODC transgenic mouse skin and tumors. For example, polyamines might indirectly alter enzymatic activity by triggering signaling events leading to posttranslational modification of HAT or HDAC enzymes. They may also provide positive or negative interference with the association of various components of HAT/HDAC protein complexes. Polyamines might also disrupt a delicate balance between HAT and HDAC enzymes through moderating sequestration and/or rates of synthesis or turnover of these proteins. In this regard, it is interesting that intrinsic HAT activity is stimulated in ODC/Ras tumors, whereas HDAC activity is not enhanced and possibly even partially repressed. Thus, the net equilibrium established between these opposing enzymatic activities may inappropriately favor acetylation. It is possible that expression of activated Ras and/or some event related to malignant conversion may serve to counteract the influence of intracellular polyamine levels on the activity of HDAC, but not of HAT, enzymes in tumorigenic cells. It may not be coincidental that similar polyamine-mediated enhancement effects on HAT function occur both in perpetually growing immortalized cells (35) and in the highly proliferative cells characteristic of ODC/Ras tumors. In contrast, normal keratinocytes terminally differentiate after very few cell divisions. This intrinsic difference in cellular programming may explain the opposite effects on histone acetylation observed in labeled primary keratinocytes as compared with cell lines (35) . The abnormally high HAT activity measured in ODC skin and tumors may translate to increased acetylation of histones or transcriptional regulatory proteins (58) , setting the stage for altered expression of at least a subset of genes. We are currently working to identify specific HAT and HDAC enzymes and gene promoters that are targets of polyamine modulation, particularly those implicated in promoting tumor development. This should permit the characterization of the various mechanisms by which polyamines regulate acetylation of nucleosomes and their respective roles in the neoplastic process.

Our studies have already revealed p300/CBP as a target of polyamine regulation. Because p300 and CBP are crucial to the integration of many diverse signaling pathways involved in controlling proliferation, differentiation, response to cell damage, and apoptosis, regulation of their function must be very tightly controlled. In fact, levels of p300/CBP appear to be rate limiting in the cell (59 , 60) . Thus, it seems likely that even relatively small perturbations in inherent p300/CBP HAT activity, such as that promoted by unregulated expression of ODC, could have major impact on cell function. Although elevated in ODC transgenic mouse skin, p300/CBP HAT activity does not appear to contribute substantially to the high HAT activity intrinsic to ODC/Ras tumors. However, effects of polyamines on CBP/p300 coactivation of even one or two critical genes may be sufficient to perturb cell homeostasis. Furthermore, CBP and p300 acetylate a variety of nonhistone proteins, including transcriptional regulators, resulting in diverse functional consequences (58) . Thus, proteins other than histones may be targets of the increased acetyltransferase activity associated with p300/CBP in K6/ODC skin. Either scenario might predispose a cell to the downstream effects of the histone H4-specific HAT activity, setting the stage for tumor development.

Although the functional significance of the specificity for Lys-12 exhibited by the HAT activity in ODC-overexpressing skin and tumors has yet to be determined, it is interesting that Lys-12 is reportedly consistently underused in monoacetylated histone H4 in several mammalian cell types, yet is frequently used in the more highly acetylated H4 isoforms typically associated with actively transcribed DNA (36) . Thus, the increased HAT activity in ODC-transgenic skin and tumors may reflect hyperacetylation and enhanced transcription at localized regions of the genome. However, perhaps of equal significance is the observation that Lys-12 is acetylated much less frequently than Lys-5, Lys-8, and Lys-16 in euchromatin, whereas enrichment of acetylated Lys-12 is a distinguishing feature of transcriptionally silent heterochromatin in yeast and higher eukaryotes (37 , 38) . The homologues PCAF and GCN5, the substrate preferences of which do not include Lys-12 (46 , 47) , as well as CBP/p300, as already discussed, can be ruled out as major contributors to the histone H4-specific HAT activity predominant in ODC/Ras tumors. Notably, the HAT B class enzymes, originally classified by their presence in the cytoplasm, are characterized by a distinct specificity for Lys-12 (and in some cases, Lys-5; 61 , 62 ). Recently, the HAT B enzyme HAT1 has been detected in the nucleus (62 , 63) and determined to be required for telomeric silencing, mediated solely through Lys-12 (39) . It is thought that acetylation of Lys-12 in histone H4 facilitates binding of silencing proteins that propagate formation of a specialized transcriptionally repressive chromatin structure (38 , 39) . Thus, the distinct preference for Lys-12 characteristic of ODC skin and tumors may be indicative of the remodeling of localized regions of chromatin into a conformation that is prohibitive to gene transcription. Given the high frequency with which ODC expression is up-regulated in human tumors, it is intriguing to speculate that improper acetylation of Lys-12 in histone H4 may be a hallmark of epithelial tumor development.

The relatively modest changes in overall acetylation of histones observed in K6/ODC skin suggest that elevated intracellular levels of polyamines do not have major impact on the acetylation of nucleosomal histones in bulk chromatin. Because transcriptionally active chromatin comprises a very small percentage of the total genome, any localized effects of polyamines on the acetylation of histones in nucleosomes bound to gene promoters would not be readily detected within the context of bulk chromatin. Ultimately, whether polyamines exert positive, negative, or no influence at all on the acetylation of histones at a given gene promoter will be determined, at least in part, by the composition of the specific regulatory complexes that are recruited (9) , as well as the subnuclear compartment in which the gene resides (64 , 65) . Indeed, our evidence for multiple mechanisms by which polyamines influence histone acetylation implies the potential for polyamines to mediate decreased acetylation of nucleosomal histones at one gene promoter while stimulating increased acetylation at another promoter, resulting in different transcriptional outcomes for those genes. Within the context of tumorigenesis, ODC overexpression might result in transcriptional repression of one or more genes critical for controlling cell proliferation, while simultaneously activating transcription of genes involved in sustaining tumor growth and invasiveness. The future challenge will be to elucidate the specific effects of polyamines on HAT and HDAC function and to determine the relative contribution of these various effects to the pathogenesis of cancer.


   ACKNOWLEDGMENTS
 
We thank Dr. Thomas O’Brien for critical review of the manuscript and Judith Mostochuk and Loretta Rossino for providing technical and editorial assistance, respectively.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grants CA 75756 (to C. A. H.) and CA 70739 (to S. K. G.) from the National Cancer Institute. Back

2 To whom requests for reprints should be addressed, at Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096. Phone: (610) 645-8429; Fax: (610) 645-2205; E-mail: gilmours{at}mlhs.org Back

3 The abbreviations used are: HAT, histone acetyltransferase; HDAC, histone deacetylase; ODC, ornithine decarboxylase; DFMO, {alpha}-difluoromethylornithine; PCAF, p300/CBP-associated-factor; CBP, CREB-binding protein. Back

Received 7/11/01. Accepted 11/ 1/01.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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