The Journal of Nutrition Vol. 127 No. 1 January 1997, pp. 18-24
Copyright ©1997 by the American Society for Nutritional Sciences

Butyrate Alters Activity of Specific cAMP-Receptor Proteins in a Transgenic Mouse Colonic Cell Line^1,2

Harold M. Aukema³, Laurie A. Davidson, Barbara C. Pence^*, Yi-Hai Jiang, Joanne R. Lupton, and Robert S. Chapkin⁴

Faculty of Nutrition, Molecular and Cell Biology Group, Texas A & M University, College Station, TX, 77843-2471 and ^* Department of Pathology, Texas Tech University Health Science Center, Lubbock, TX, 79430

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

FOOTNOTES

ACKNOWLEDGMENT

LITERATURE CITED

ABSTRACT

There is great interest in utilizing butyrate as a chemotherapeutic agent. To elucidate its mechanism of action, the effect of butyrate on cAMP receptor protein kinase (PKA) activity in young adult mouse colon (YAMC) cells isolated from transgenic mice bearing a temperature sensitive mutation of the SV40 large T antigen gene was investigated. Conditionally immortalized cultures were plated at the permissive temperature (33°C) or growth arrested by incubation at the nonpermissive temperature (39°C). In addition, cells were incubated at 33°C with or without 1 mmol/L butyrate for 24 h. Butyrate treatment reduced cell proliferation by 28% and enhanced apoptosis by 350% compared with cultures not exposed to butyrate. The PKA type I/II isozyme activity ratio was lower (P < 0.05) in cells incubated with butyrate. The relative level of PKA I isozyme was higher in proliferating cells at 33°C (63% of total PKA), while the relative level of PKA II was higher in nonproliferating cells undergoing apoptosis at 39°C (59% of total PKA). Neither incubation conditions (33 vs. 39°C) nor butyrate treatment altered total PKA activity. When YAMC cells were incubated with 8-Cl-cAMP, an activator of PKA II, growth was markedly inhibited in cells at both temperatures. Consistent with in vitro data, increased PKA I isozyme levels were associated with dysregulated growth in vivo. Specifically, the relative level of PKA I isozyme was three- to fivefold higher in rat colonic tumors compared with normal nontransformed colonic mucosa. These data indicate that the biological effects of butyrate on colonocyte proliferation and apoptosis are associated with changes in PKA isozyme-dependent signal transduction, and the YAMC cell line is a relevant model to examine the molecular mechanisms by which dietary-derived factors affect relative cancer risk.

INTRODUCTION

Epidemiological studies generally support a protective role of dietary fibers in colon carcinogenesis (Heilbrun et al. 1989, Trock et al. 1990). Experiments in the murine model using a variety of fibers and carcinogens, however, have produced conflicting results (Jacobs and Lupton 1986, Rogers and Nauss 1984). Because fibers are a heterogeneous group of compounds with different physicochemical properties, this is not unexpected. The poorly fermentable fibers such as wheat bran and cellulose generally protect against experimentally induced colon cancer (Barbolt et al. 1978, Heitman et al. 1989, Watanabe et al. 1979, Wilson et al. 1977), whereas the more fermentable fibers such as pectin, guar gum, oat bran, agar and carageenan do not protect and may actually enhance tumorigenesis (Bauer et al. 1981, Jacobs and Lupton 1986, Watanabe et al. 1978).

One of the products of fiber fermentation that is believed to play an important role in the colon is the four carbon, short-chain fatty acid, butyrate. As fiber is fermented, CO₂, H₂, CH₄ and short-chain fatty acids (acetate, propionate, butyrate) are produced (McNeil 1984), with the short-chain fatty acids constituting the primary anions in the stool. Short-chain fatty acids, including butyrate, are stimulants of normal colonic cell proliferation in vivo (Newmark and Lupton 1990, Sakata 1987) and normal colonic mucosa in vitro (Scheppach et al. 1992). This is noteworthy because an increase in cell proliferation may be tumor enhancing (Farber 1984, Romagnoli et al. 1984, Williamson and Rainey 1984). In contrast to effects in vivo, butyrate has been shown to inhibit growth in a variety of human colon cancer cell lines (Chung et al. 1985, Lupton 1995, Whitehead et al. 1986) and to enhance apoptosis (Hague et al. 1993, Heerdt et al. 1994). In view of the observations involving malignant transformed cell lines, there is great interest in utilizing butyrate as a chemotherapeutic and chemopreventive agent (Newmark et al. 1994, Rephaleli et al. 1991). However, the molecular mechanisms responsible for the effects of butyrate on colonocyte biology are not known.

The cAMP signaling pathway has been implicated in the regulation of colonocyte growth and tumor development (Aukema et al. 1994, DeRubertis et al. 1976, Stevens and Loven 1980). Cyclic AMP is involved in the regulation of a number of cellular processes, including cell growth, differentiation and apoptosis (Cohen and Hardie 1991, Roesler et al. 1988). Other than the direct regulation of selection channels, the only mechanism by which cAMP exerts its effects in eukaryotes is the activation of cAMP-dependent protein kinase (cAMP receptor protein kinase; ATP:protein phosphotransferase, PKA, EC 2.7.1.37). PKA type I and type II isozymes, containing unique R subunits, RI and RII, respectively, were originally distinguished by their elution patterns from DEAE-cellulose columns (Taylor et al. 1990). Presently, four isozymes of the R subunits (RI, RI, RII, RII), three isozymes of the C subunits (C, C, C), and splice variants of the C subunits have been identified (Scott 1991). The activated C subunits modulate cellular events both at the post-translational level by phosphorylation of cellular substrates and at the transcriptional level by phosphorylation of transcription factors which interact with regulatory elements of cAMP-responsive genes (Roesler et al. 1988, Taylor et al. 1990).

Most tissues contain a mixture of PKA type I and II isozymes (Cho-Chung 1993). It has been hypothesized that the selective expression of these isozymes is a critical component in the control of cell growth and differentiation (Cho-Chung 1993). For example, in cultured cells, the RI/RII ratio increases with cell growth and decreases with differentiation (Cho-Chung 1993, DeRubertis and Craven 1980). Site-selective analogs and antisense oligodeoxynucleotides, which deplete the RI subunit, have been used to inhibit the growth of human cancer cells (Bradbury et al. 1994, Cho-Chung 1993, Yokozaki et al. 1993). Currently, some of these agents are being tested in humans in phase I clinical studies by the National Cancer Institute (Cho-Chung 1993). As related to cancer, colonic tumors overexpress RI when compared with uninvolved colonic mucosa (Bradbury et al. 1994, DeRubertis et al. 1980). More specifically, in normal rat colonic mucosa, 90-95% of PKA activity is associated with the type II isozyme, whereas in rat tumors only 65-75% of PKA activity is associated with the type II isozyme (Aukema et al. 1994).

We have investigated the effect of butyrate on PKA activity in the young adult mouse colon (YAMC) cell line which was isolated from transgenic mice bearing a temperature-sensitive mutation of the SV40 large T antigen gene (Whitehead et al. 1993). At the permissive temperature (33°C), the SV40 large T antigen gene product is functional; cells are immortalized and can be cultured indefinitely. When the gene product is inactivated at the nonpermissive temperature (39°C), however, these cells stop proliferating within 2 d and die after 5-7 d, similar to normal colonocytes in vivo.

---

MATERIALS AND METHODS

Materials. Aprotinin, soybean trypsin inhibitor, leupeptin, pepstatin, 4-(2-aminoethyl) benzene-sulfonyl fluoride, 3-isobutyl-1-methyl-xanthine, sodium fluoride, EDTA, EGTA, Triton X-100 and p-nitrophenol phosphate were purchased from Sigma Chemical (St. Louis, MO). ATP was obtained from Boehringer Mannheim, Indianapolis, IN) and 8-Cl-cAMP was from P-L Biochemicals (Milwaukee, WI). Fetal bovine serum was from Hyclone (Logan, UT). [-³²P]ATP (111 TBq/mmol) was purchased from Du Pont New England Nuclear (Boston, MA). Cell culture media were from Mediatech (Herndon, VA) and media supplements from Collaborative Biochemical Research (Bedford, MA). Mouse recombinant -interferon was purchased from Genzyme (Cambridge, MA).

Cell culture. YAMC cells were obtained from R. H. Whitehead, Ludwig Cancer Institute, Melbourne, Australia (Whitehead et al. 1993). YAMC cells (passages 10-14) were cultured in RPMI 1640 (Mediatech) plus 5% fetal calf serum, 1% insulin/transferrin/selenium, 100,000 U/L penicillin, and 100 mg/L streptomycin. Cultures also contained 5000 U/L of recombinant -interferon which is required because the SV40 large T gene is under an interferon-inducible promoter. Cells were cultured under permissive (33°C) or nonpermissive (39°C) conditions. All cultures were mycoplasma free as determined by a PCR-based ELISA assay (Boehringer Mannheim). The expression of SV40 large T antigen protein was determined by immunoblotting as previously described (Davidson et al. 1994), using a mouse polyclonal antibody (Oncogene Science, Uniondale, NY). To examine the effects of butyrate on YAMC proliferation, differentiation and apoptosis, cells were cultured at 33°C.

Cell proliferation. Cell proliferation was determined by counting trypsinized cells with a hemacytometer or by the MTS Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI).

Cell differentiation. Alkaline phosphatase was measured using p-nitrophenol phosphate as substrate (Lowry et al. 1954). The assay mixture consisted of 50 mmol/L glycine buffer (pH 9.2), 5 mmol/L MgCl₂, 1 mmol/L ZnSO₄ and 18 mmol/L of substrate in a final volume of 195 µL.

Apoptosis. The cellular DNA fragmentation ELISA assay (Boehringer Mannheim) was used to quantify the level of apoptosis in YAMC cells. Values were normalized to the number of adherent cells per dish. For this assay, 30,000 cells were seeded into 35-mm dishes at 33°C overnight. The following day, cells were refed with media containing 10 µmol/L bromodeoxyuridine and either maintained at 33°C or transferred to 39°C (nonpermissive temperature). Media were replaced each day for 72 h, but contained bromodeoxyuridine only for the first 24 h. For butyrate experiments, 33°C cultures were incubated with 1 mmol/L butyrate for 24 h. Floating and adherent cells were harvested to measure levels of DNA fragmentation. Cells were lysed using reagents supplied and centrifuged at 250 × g to sediment the intact nuclei. Supernatants containing the low molecular weight DNA were subsequently analyzed by ELISA.

DNA laddering was determined using the method of White et al. (1984). Briefly, medium containing floating cells or adherent cells scraped into PBS was centrifuged at 200 × g for 3 min. The pellet was then dried and stored at 80° C. Cells were lysed by incubating with 1 mL (per 2 × 10⁶ cells) lysing solution containing 10 mmol/L Tris-HCl pH (7.9), 5 mmol/L EDTA, 100 mmol/L NaCl, 0.5% SDS, 1 mg/mL proteinase K (Ambion, Austin, TX) for 2 h at 37°C. After adding 225 µL of 5 mol/L NaCl, samples were incubated at 4°C overnight and centrifuged at 60,000 × g for 20 min. The resulting supernatant was extracted with an equal volume of phenol/chloroform/ iso-amyl alcohol (24:25:1, v/v/v), and DNA was precipitated with 2.5 volumes of ethanol. The dried pellet was resuspended in 20 µL of water and 1 µL of RNase A (10 g/L, 5-Prime-3-Prime, Boulder, CO), and incubated at 37°C for 30 min. Samples were run on 2% agarose gels and stained with ethidium bromide. Acridine orange staining of apoptotic bodies was performed on separate samples as previously described (McConkey et al. 1988).

Protein kinase A activity. Protein extracts were obtained by scraping adherent cells into homogenization buffer containing 50 mmol/L Tris-HCl (pH 7.2), 250 mmol/L sucrose, 1% Triton X-100, 50 mmol/L NaF, 2 mmol/L EDTA, 1 mmol/L EGTA, 10 mmol/L 2-mercaptoethanol, 1 mg/L soybean trypsin inhibitor, 35 mg/L 4-(2-aminoethyl) benzene-sulfonyl fluoride, and 25 mg/L of aprotinin, pepstatin, and leupeptin. After sonicating, samples were centrifuged at 13,000 × g at 4°C for 5 min. All extractions were carried out on ice. YAMC cell PKA activity and isoform distribution were determined as described by Aukema et al (1994). Briefly, PKA isoforms were separated by anion-exchange chromatography, and PKA activity of pre-column fractions or eluted fractions was measured by the phosphorylation of a PKA specific substrate (Kemptide).

Normal nontransformed rat colonic mucosa for PKA isoform analysis was obtained and processed as described previously (Aukema et al. 1994). Colonic tumors were obtained from carcinogen-injected rats (Pence et al. 1995). Briefly, frozen tumors were pulverized in liquid nitrogen, mucosa was scraped from normal rat colons and homogenized in buffer, and PKA activity determined. Protein extracts were centrifuged at 100,000 × g for 30 min at 4°C in homogenization buffer without or with 1% Triton X-100 to obtain cytosolic and particulate extracts, respectively. PKA type I and type II were separated on DEAE-cellulose columns, and PKA activity determined in the cytosolic and particulate fractions (Aukema et al. 1994).

Statistical analysis. Effects of butyrate on various parameters were analyzed by ANOVA using SAS (SAS 1985). Differences between means were separated by the least-squares means test. Data represent a minimum of two separate experiments, each performed in triplicate (n = 2).

---

RESULTS

Cell proliferation. Cells grown at the permissive temperature proliferated rapidly (Fig. 1). When transferred to the nonpermissive temperature, cell number decreased after 4 d. Similar results were observed for cell protein and [³H]thymidine uptake (data not shown). YAMC cells grown at the permissive temperature (33°C) expressed the SV40 large T antigen protein, whereas cells grown at the nonpermissive temperature (39°C) had significantly reduced (P < 0.05) levels (Fig. 2).

---

Fig. 1. Effect of cell culture temperature on young adult mouse colon (YAMC) cell proliferation. Cells were plated in 35-mm dishes and cultured at the permissive temperature (33°C) for 2 d. On d 2, dishes were incubated at either 33°C or transferred to the nonpermissive temperature (39°C) and adherent cell number, mean ± SEM determined. Data are from three separate experiments, each performed in triplicate (n = 3).
[View Larger Version of this Image (9K GIF file)]

---

---

Fig. 2. Relative levels of SV40 large T antigen in adherent young adult mouse colon (YAMC) cells cultured at the permissive (33°C) and nonpermissive (39°C) temperatures as determined by immunoblotting and densitometric scanning. Thirty micrograms of protein from YAMC cell extracts was used for SDS-PAGE (4-12% gels). Values represent means ± SEM from two separate experiments. Different letters indicate that means are significantly different, P < 0.05.
[View Larger Version of this Image (44K GIF file)]

---

To determine the effect of butyrate on YAMC cell proliferation, 33°C cultures were grown in the presence of 1 mmol/L butyrate for 24 h. Cell number was significantly lower (P < 0.05) after 24 h in cultures treated with butyrate, 89,260 ± 2,200 vs. 64,000 ± 3,400. Data are from four separate experiments, each performed in triplicate (n = 4). The effect of butyrate (0.1-10 mmol/L) on cell number was dose dependent as confirmed by the MTS cell proliferation assay (data not shown). The effects of butyrate on cell proliferation were not the result of alterations in the SV40 gene product, because butyrate did not affect the expression of SV40 large T antigen (Fig. 3).

---

Fig. 3. Effect of 1 mmol/L butyrate (But) on the relative levels of SV40 large T antigen in adherent young adult mouse colon (YAMC) cells cultured at 33 and 39°C. Thirty micrograms of protein from YAMC cell extracts, or rat colonic mucosa as the negative control, was used for SDS-PAGE (4-12% gels). From left to right; lane 1, 33°C But; lane 2, 33°C + But; lane 3, 39°C But; lane 4, 39°C + But; lane 5, rat colonic mucosa. Arrow indicates an immunoreactive band at 94 kDa. Data are representative of two separate experiments.
[View Larger Version of this Image (68K GIF file)]

---

Cell differentiation. To examine whether adherent cells cultured at the nonpermissive temperature expressed a differentiated phenotype, alkaline phosphatase activity was determined. Data were derived from cells cultured at 33°C for 2-4 d and from cells at 39°C for 1-5 d (n = 3). Although there were large differences in the rates of proliferation in cells grown at 33°C compared with 39°C, the levels of alkaline phosphatase activity were not different, 3.76 ± 0.38 vs. 3.66 ± 0.27 units/µg protein, respectively. In comparison, butyrate had no significant effect on alkaline phosphatase activity in adherent cells grown at the permissive temperature (data not shown). These data indicate that neither culture temperature nor butyrate dosing altered the differentiation state of these cells.

Apoptosis. To examine whether growth arrest at the nonpermissive temperature was associated with enhanced apoptosis, cellular DNA fragmentation was determined by electrophoretic laddering and acridine orange staining. Using these methodologies, apoptosis was detected in the floating (nonadherent) cells at both temperatures by DNA laddering (Fig. 4). DNA fragmentation was not detected in adherent cells at either temperature when using DNA laddering. Acridine orange staining confirmed that very few cells were undergoing nuclear condensation and fragmentation in the adherent cell populations. Because these assays are not quantitative, the level of apoptosis was measured by cellular DNA fragmentation ELISA. Using this method, the level of apoptosis in the adherent cell population was very low, <1% of that in the floating cells on a cell number basis. In YAMC cells cultured at both the permissive and nonpermissive temperatures, the level of apoptosis on a per cell number basis did not change significantly over time. However, apoptosis was greater (P < 0.05) in nonadherent cells grown at 39°C compared with those at 33°C at all time points examined (Table 1). When YAMC cells were exposed to 1 mmol/L butyrate for 24 h at 33°C, the apoptotic index was also increased (P < 0.05); 33°C without butyrate, 0.76 ± 0.20 and 33°C with butyrate, 2.71 ± 0.56. Values represent mean absorbance at 450 nm ± SEM divided by total adherent cells per dish (×10⁵). Data are from our separate experiments, each performed in triplicate (n = 4). Overall, the percentage of shed cells exhibiting an apoptotic morphology was not altered by temperature or butryate treatment.

---

Fig. 4. Effect of cell culture temperature on young adult mouse colon (YAMC) cell apoptosis. DNA laddering was measured in floating and adherent YAMC cells cultured at the permissive (33°C) and nonpermissive (39°C) temperatures. Floating cells were obtained from cultures at 39°C for 2 d (lane 1) and 4 d (lane 4), or at 33°C for 3 d (lane 7) and 5 d (lane 8). Adherent cells were scraped from plates cultured at 39°C for 2 d (lane 2) and 4 d (lane 3), or at 33°C for 3 d (lane 5) and 5 d (lane 6). DNA markers are shown in lane 9. Data are representative of two separate experiments.
[View Larger Version of this Image (99K GIF file)]

---

Table 1. Effect of cell culture temperature on young adult mouse colon (YAMC) cell apoptosis1,2 [View Table]

Protein kinase A isozyme activity. PKA signal transduction may play an important role in the regulation of colonocyte proliferation (Aukema et al. 1994, Bradbury et al. 1994). To determine the importance of this signaling pathway in YAMC cells, the relative activity levels of PKA isozymes were examined in adherent cells grown at 33°C compared with 39°C. In rapidly proliferating cells at the permissive temperature, PKA I was the predominant isozyme (63% of total PKA). When these cells were transferred to 39°C, however, PKA II was present at a higher level (59% of total PKA) than PKA I (Fig. 5). The total PKA activity in cells cultured at 33°C for 2-3 d compared with cells cultured at 39°C for 1-5 d, however, was not significantly different (Fig. 5, inset). Butyrate also induced changes in the relative activity levels of PKA isozymes. Cells dosed at the permissive temperature expressed lower activity levels of PKA I (Fig. 6). No changes in total PKA activity were observed (Fig. 6, inset), indicating that butyrate effects on PKA I isozyme activity occurred at the expense of changes in PKA II.

---

Fig. 5. Relative levels of protein kinase A (PKA) isozymes in adherent young adult mouse colon (YAMC) cells cultured at the permissive temperature (33°C) for 2-3 d or the nonpermissive temperature (39°C) for 2-5 d. *Significantly different (P < 0.05) from the corresponding PKA isozyme value obtained from cells cultured at 33°C. Inset: total PKA activity [pmol of phosphate transferred/(min·µg protein)] in adherent YAMC cells cultured at the permissive temperature (33°C) for 2-3 d or the nonpermissive temperature (39°C) for 1-5 d. Data are from three separate experiments, each performed in triplicate (n = 3).
[View Larger Version of this Image (34K GIF file)]

---

---

Fig. 6. Effect of 1 mmol/L butyrate (But) on the relative level of protein kinase A (PKA) type I isozyme in adherent young adult mouse colon (YAMC) cells cultured at 33°C. Cells were exposed to butyrate for 24 h as described in Materials and Methods. Bars having different letters are different (P < 0.05) from each other. Inset: effect of 1 mmol/L butyrate on total PKA activity [pmol phosphate transferred/(min·µg protein)] in adherent YAMC cells cultured at 33°C. Data are from four separate experiments, each performed in triplicate (n = 4).
[View Larger Version of this Image (54K GIF file)]

---

Effect of 8-Cl-cAMP on YAMC cell proliferation. The importance of the PKA signaling pathway in the control of cell growth was also examined by using cAMP analogs which selectively activate the PKA II isozyme. These analogs have been shown to inhibit growth in a variety of transformed cell lines (Cho-Chung 1993, Yokozaki et al. 1993). Subsequently, when YAMC cells were incubated with 8-Cl-cAMP, growth was significantly inhibited (P < 0.05) in cells at both temperatures, with inhibition being greater in the cells cultured at the nonpermissive temperature (Fig. 7). Cell proliferation was determined by measuring absorbance at 490 nm using an MTS-based assay (n = 2) and was verified by cell counting. The addition of butyrate to YAMC cells exposed to 8-Cl-cAMP did not influence the effect of this analog on cell growth (data not shown).

---

Fig. 7. Effect of 8-Cl-cAMP (50 µmol/L) on young adult mouse colon (YAMC) cell proliferation. Cells were cultured at the permissive temperature (33°C) and the nonpermissive temperature (39°C). All values are different (P < 0.05) from time 0. Control cultures contained no 8-Cl-cAMP. Values represent means ± SEM from two separate experiments, each performed in triplicate (n = 2).
[View Larger Version of this Image (10K GIF file)]

---

Protein kinase A isozyme levels in rat colonic mucosa and colonic tumors. To determine the relevance of the YAMC PKA data, isozyme levels were examined in normal nontransformed rat colonic mucosa and rat colonic tumors. Total PKA activity for each tissue preparation was as follows: tumor cytosol, 5.17 ± 0.87 pmol phosphate transferred (min·µg protein); tumor membrane, 5.66 ± 1.53; normal cytosol, 5.54 ± 0.62; and normal membrane, 4.50 ± 0.79. In normal colonic mucosa, PKA I isozyme comprised 11 and 5% of total PKA activity in the cytosolic and particulate fractions, respectively (Fig. 8). In comparison, the relative activity level of PKA I in colonic tumors was three- to fivefold higher, at levels of 36 and 25% of total PKA in the cytosolic and particulate fractions, respectively (Fig. 8). Hence, malignant transformation was associated with higher relative activity levels of PKA I both in colonic tumors in vivo and in rapidly proliferating YAMC cells in vitro. Normal rates of proliferation and apoptosis in nontransformed colonic mucosa in vivo were associated with higher activity levels of PKA II, comparable to YAMC cells cultured at 39°C in vitro.

---

Fig. 8. Relative levels of cyclic AMP-dependent protein kinase (PKA) isozymes in normal nontransformed rat colonic mucosa and rat colon carcinomas. Particulate, 100,000 × g pellet, and cytosol, 100,000 × g supernatant, were assayed for PKA isozyme activity as described in Materials and Methods. *Different (P < 0.05) from corresponding PKA isozyme values obtained from normal nontransformed tissue. Values represent means ± SEM, n = 6-10 mice.
[View Larger Version of this Image (49K GIF file)]

---

---

DISCUSSION

Because normal nontransformed colonocytes are difficult to culture for an extended period of time, transformed cell lines have typically been used. However, to study the intracellular signaling pathways which regulate normal colonocyte proliferation and programmed cell death, a more relevant model system is required. In this study, we have demonstrated that the YAMC cell line can be utilized to investigate colonocyte PKA signal transduction in vitro. YAMC cells cultured at a nonpermissive temperature of 39°C resemble normal colonocytes in vivo because they have a limited lifespan. The immortalized cells at 33°C resemble transformed cells, because they can be cultured indefinitely. Cells transferred from the permissive temperature to the nonpermissive temperature cease proliferation, with cell number decreasing after 4 d. Reduction of cell growth, however, does not appear to coincide with differentiation, as measured by alkaline phosphatase activity. Apoptosis occurs in cells grown at either temperature, but at substantial levels only in nonadherent, floating cells. Acridine orange staining showed that most floating cells were undergoing nuclear condensation characteristic of apoptosis (data not shown). Quantification of DNA fragmentation by ELISA indicated a significantly higher (P < 0.05) level of apoptosis in cells grown at the nonpermissive temperature compared with those cultured at the permissive temperature. Therefore, apoptosis plays an important role in the turnover of colonocytes both in vitro and in vivo (Potten et al. 1992).

Epidemiological studies have linked fiber to the prevention of colon cancer (Heilbrun 1989, Trock et al. 1990). One product of fiber fermentation in the colon is butyrate, and, unfortunately, the molecular mechanisms by which this short-chain fatty acid exerts its biological effects have not been fully resolved (Hague et al. 1993). Butyrate analogs are currently being investigated as potential antineoplastic agents (Newmark et al. 1994, Rephaeli et al. 1991). In this study, we report that butyrate inhibited YAMC cell proliferation and enhanced apoptosis when cultured at the permissive temperature. These results are consistent with previous experiments using colonic carcinoma cell lines (Chung et al. 1985, Heerdt et al. 1994, Whitehead et al. 1986). The effects of butyrate are noteworthy, because cancer is a disease characterized by the accumulation of cells, and the abnormal accumulation of cells can result from either increased proliferation or the failure of cells to undergo apoptosis in response to an appropriate signal (Farber 1995, Potten et al. 1992). Interestingly, the percentage of shed cells exhibiting an apoptotic morphology was not altered by butyrate treatment, which is consistent with previous experiments using human colonic carcinoma cell lines (Heerdt et al. 1994). The fact that the bulk of cells shed into the media exhibited an apoptotic phenotype implies that once cells lose contact with the underlying matrix they undergo programmed cell death (Ruoslahti and Reed 1994). Therefore, it is possible that butyrate initiates apoptosis in adherent cell populations by disrupting interactions between cells and the extracellular matrix (Frisch and Francis 1994, Heerdt et al. 1994). Further experiments are required to test this hypothesis.

PKA type I is considered to enhance cell proliferation and transformation, while the type II isozyme regulates inhibition of cell growth (Cho-Chung 1993). In rapidly proliferating tissues, such as during embryonal development and in tumors, the proportion of PKA I is increased compared with that of normal nontransformed tissue (Bradbury et al. 1994, Cho-Chung 1993). Although total PKA activity was not different in YAMC cells grown at 33°C compared with 39°C, the relative activity levels of the PKA isoforms were significantly altered. The relative activity level of PKA I was higher in the rapidly growing cells at the permissive temperature, whereas growth-restricted cells had a higher relative activity of PKA II. In addition, compared with normal nontransformed colonic mucosa, rat colonic tumors expressed higher activity of PKA I relative to the PKA II isozyme, which is consistent with data from tumors isolated from lung, mammary, gastric and colon tissues (Bradbury et al. 1994, Cho-Chung 1993). Therefore, with regard to PKA isozyme activity, YAMC cells grown at the nonpermissive (39°C) and permissive (33°C) temperatures are somewhat representative of in vivo normal nontransformed mucosa and colonic tumors, respectively.

We have postulated that the protective effects of butyrate may be linked to alterations in cAMP-receptor proteins. Consistent with its ability to inhibit cell proliferation and potentiate apoptosis, butyrate induced a modest but significant decrease in the PKA I/II activity ratio. In addition, cells incubated with 8-Cl-cAMP, which increases the relative activity levels of PKA II, exibited decreased proliferation when cultured at either the permissive or nonpermissive temperature. This is significant because there is considerable interest in the use of cAMP analogs as chemotherapeutic agents (Cho-Chung 1993).

In conclusion, the in vitro and in vivo data in this study suggest that the enhancement of PKA I activity is associated with increased colonic proliferation and malignant transformation, whereas PKA II activity is associated with reduced proliferation and elevated levels of apoptosis. This work also supports the hypothesis that butyrate-induced effects on colonic cell proliferation and apoptosis may be mediated in part by biochemical alterations in PKA signal transduction.

FOOTNOTES

Manuscript received 22 July 1996. Initial reviews completed 21 August 1996. Revision accepted 9 September 1996.

ACKNOWLEDGMENT

We are grateful to R. H. Whitehead for supplying the YAMC cell line and for helpful discussions.

LITERATURE CITED

• Aukema H. M., Davidson L. A., Derr J. N., Lupton J. R., Chapkin R. S. Diet modulation of rat colonic cAMP-dependent protein kinase activity. Biochim. Biophys. Acta 1994; 1224:51-60 [Medline]
• Barbolt T. A., Abraham R. The effect of bran in dimethylhydrazine-induced colon carcinogenesis in the rat. Proc. Soc. Exp. Biol. Med. 1978; 157:656-659 [Medline]
• Bauer H. G., Asp N.-G., Dahlqvist A., Fredlung P. E., Nyman M., Oste R. Effect of two kinds of pectin and guar gum on 1,2-dimethylhydrazine initiation of colon tumors and on fecal -glucuronidase activity in the rat. Cancer Res. 1981; 41:2518-1523 [Abstract/Free Full Text]
• Bradbury A. W., Carter D. C., Miller W. R., Cho-Chung Y. S., Clair T. Protein kinase A (PK-A) regulatory subunit expression in colorectal cancer and related mucosa Br. J. Cancer 1994; 69:738-742
• Cho-Chung Y. S. Differentiation therapy of cancer targeting the RI regulatory subunit of cAMP-dependent protein kinase (review). Int. J. Oncol. 1993; 3:141-148
• Chung Y. S., Song I. S., Erickson R. H., Sleisenger M. H., Kim Y. S. Effect of growth and sodium butyrate on brush border membrane-associated hydrolases in human colorectal cancer cell lines. Cancer Res. 1985; 45:2976-2982 [Abstract/Free Full Text]
• Cohen P., Hardie D. G. The actions of cyclic AMP on biosynthetic processes are mediated indirectly by cyclic AMP-dependent protein kinase. Biochim. Biophys Acta 1991; 1004:292-299
• Davidson L. A., Jiang Y. H., Derr J. N., Aukema H. M., Lupton J. R., Chapkin R. S. Protein kinase C isoforms in human and rat colonic mucosa. Arch. Biochem. Biophys. 1994; 312:547-553 [Medline]
• DeRubertis F. R., Chayoth R., Field J. B. The content and metabolism of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate in adenocarcinoma of the human colon. J. Clin. Invest. 1976; 57:641-649
• DeRubertis F. R., Craven P. A. Early alterations in rat colonic mucosal cyclic nucleotide metabolism and protein kinase activity induced by 1,2-dimethylhydrazine. Cancer Res. 1980; 40:4589-4598 [Abstract/Free Full Text]
• Farber E. Cellular biochemistry of the stepwise development of cancer with chemicals. Cancer Res. 1984; 44:5463-5474 [Abstract/Free Full Text]
• Farber E. Cell proliferation as a major risk factor for cancer: a concept of doubtful validity. Cancer Res. 1995; 55:3759-3762 [Free Full Text]
• Frisch S. M., Francis H. Disruption of epithelial cell-matrix interactions induces apoptosis. J. Cell. Biol. 1994; 124:619-626 [Abstract/Free Full Text]
• Hague A., Mannign A. M., Hanlon K. A., Huschtscha L. I., Hart D., Paraskeva C. Sodium butyrate induces apoptosis in human colonic tumour cell lines in a p53-independent pathway: implications for the possible role of dietary fibre in the prevention of large-bowel cancer. Int. J. Cancer 1993; 55:498-505 [Medline]
• Heerdt B. G., Houston M. A., Augenlicht L. H. Potentiation by specific short-chain fatty acids of differentiation and apoptosis in human colonic carcinoma cell lines. Cancer Res. 1994; 54:3288-3294 [Abstract/Free Full Text]
• Heilbrun L. K., Nomura A., Hankin J. H., Stemmermann G. N. Diet and colorectal cancer with special reference to fiber intake. Int. J. Cancer 1989; 44:1-6 [Medline]
• Heitman D. W., Ord V. A., Hunter K. E., Cameron I. L. Effect of dietary cellulose on cell proliferation and progression of 1,2-dimethylhydrazine-induced colon carcinogenesis in rats. Cancer Res. 1989; 49:5581-5585 [Abstract/Free Full Text]
• Jacobs L. R., Lupton J. R. Relationship between colonic luminal pH, cell proliferation, and colon carcinogenesis in 1,2-dimethylhydrazine treated rats fed high fiber diets. Cancer Res. 1986; 46:1727-1734 [Abstract/Free Full Text]
• Lowry O. H., Roberts N. R., Wu M.-L., Hixon W. S., Crawford E. J. The quantitative histochemistry of brain: II. Enzyme measurements. J. Biol. Chem. 1954; 207:19-28 [Free Full Text]
• Lupton J. R. Butyrate and colonic cytokinetics: Differences between in vitro and in vivo studies. Eur. J. Cancer Prev. 1995; 4:373-378 [Medline]
• McConkey, D. J., Hartzell, P, Duddy, S. K., Hakansson, H. & Orrenius, S. (1988) 2,3,7,8-Tetrachlorodibenzo-p-dioxin kills immature thymocytes by Ca²⁺-mediated endonuclease activation. Science (Washington, DC) 242: 256-259.
• McNeil N. I. The contribution of the large intestine to energy supplies in man. Am. J. Clin. Nutr. 1984; 39:338-342 [Abstract/Free Full Text]
• Newmark H. L., Lupton J. R. Determinants and consequences of colonic luminal pH: Implications for colon cancer. Nutr. Cancer 1990; 14:161-173 [Medline]
• Newmark H. L., Lupton J. R., Young C. W. Butyrate as a differentiating agent: pharmacokinetics, analogues and current status. Cancer Lett. 1994; 78:1-5 [Medline]
• Pence B. C., Dunn D. M., Zhao C., Landers M., Wargovich M. J. Chemopreventive effects of calcium but not aspirin supplementation in cholic acid-promoted colon carcinogenesis: correlation with intermediate endpoints. Carcinogenesis 1995; 16:757-765 [Abstract/Free Full Text]
• Potten C. S., Li Y. Q., O'Connor P. J., Winton D. J. A possible explanation for the differential cancer incidence in the intestine, based on distribution of the cytotoxic efects of carcinogens in the murine large bowel. Carcinogenesis 1992; 13:2305-2312 [Abstract/Free Full Text]
• Rephaeli A., Rabizadeh E., Aviram A., Shaklai M., Ruse M., Nudelman A. Derivatives of butyric acid as potential anti-neoplastic agents. Int. J. Cancer 1991; 49:66-72 [Medline]
• Roesler W. J., Vandenbark G. R., Hanson R. W. Cyclic AMP and the induction of eukaryotic gene transcription. J. Biol. Chem. 1988; 263:9063-9066 [Free Full Text]
• Rogers, A. E. & Nauss, K. M. (1985) Contributions of laboratory animal studies of colon carcinogenesis. In: Large Bowel Cancer (Mastromarino, A. J. & Brattain, M. G., eds.), pp. 1-45. Praeger, New York, NY.
• Romagnoli P., Filipponi F., Bandettini L., Bregnola D. Increase of mitotic activity in the colonic mucosa of patients with colorectal cancer. Dis. Colon Rectum 1984; 27:305-308 [Medline]
• Ruoslahti E., Reed J. C. Achorage dependence, integrins, and apoptosis. Cell 1994; 77:477-478 [Medline]
• Sakata T. Stimulatory effect of short-chain fatty acids on epithelial cell proliferation in the rat intestine: a possible explanation for trophic effect of fermentable fiber, gut microbes and luminal trophic factors. Br. J. Nutr. 1987; 58:95-103 [Medline]
• SAS Institute Inc. (1985) SAS User's Guide. SAS Institute, Cary, NC.
• Scheppach W., Bartram P., Richter A., Richter F., Liepold H., Dusel G., Hofstetter G., Ruthlein J., Kasper H. The effect of short-chain fatty acids on the human colonic mucosa in vitro. J. Parent. Enteral Nutr. 1992; 16:43-48 [Abstract/Free Full Text]
• Scott J. D. Cyclic nucleotide-dependent protein kinases. Pharmacol. Ther. 1991; 50:123-145 [Medline]
• Stevens, R. H. & Loven, D. P. (1980) Intracellular adenosine and guanosine 3',5'-cyclic monophosphate concentrations in rat small and large bowel following single and multiple exposures to 1,2-dimethylhydrazine. Cancer Lett. 9: 151-159.
• Taylor S. S., Buechler J. A., Yomemoto W. cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. Annu. Rev. Biochem. 1990; 59:971-1005 [Medline]
• Trock B., Lanza E., Greenwald P. Dietary fiber, vegetables, and colon cancer: a critical review and meta-analysis of the epidemiologic evidence. J. Natl. Cancer Inst. 1990; 82:650-661 [Abstract/Free Full Text]
• Watanabe K., Reddy B. S., Weisburger J. H., Kritchevsky D. Effect of dietary alfalfa, pectin and wheat bran on azoxymethane- or methylnitrosourea-induced colon carcinogenesis in F344 rats. J. Natl. Cancer Inst. 1979; 63:141-145
• Watanabe K., Reddy B. S., Wong C. Q., Weisburger J. H. Effect of dietary undegraded carageenan on colon carcinogenesis in F344 rats treated with azoxymethane or methylnitrosourea. Cancer Res. 1978; 38:4427-4430 [Abstract/Free Full Text]
• White E., Grodzicker T., Stillman B. W. Mutations in the gene encoding the adenovirus early region 1B 19,000-molecular-weight tumor antigen cause the degradation of chromosomal DNA. J. Virol. 1984; 52:410-419 [Abstract/Free Full Text]
• Whitehead R. H., VanEeden P. E., Noble M. D., Ataliotis P., Jat P. S. Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2K^b-tsA58 transgenic mice. Proc. Nat. Acad. Sci. U.S.A. 1993; 90:587-591 [Abstract/Free Full Text]
• Whitehead R. H., Young G. P., Bhatal P. S. Effects of short chain fatty acids on a new human colon carcinoma cell line (LIM1215). Gut 1986; 27:1457-1463 [Abstract/Free Full Text]
• Williamson, R.C.N. & Rainey, J. R. (1984) The relationship between intestinal hyperplasia and carcinogenesis. Scand. J. Gastroenterol. 19 (Suppl. 104): 57-76.
• Wilson R. B., Hutcheson D. P., Wideman L. Dimethylhydrazine-induced colon tumors in rats fed diets containing beef fat and corn oil with and without wheat bran. Am. J. Clin. Nutr. 1977; 30:176-181 [Abstract/Free Full Text]
• Yokozaki H., Budillon A., Tortora G., Meissner S., Beaucage S. L., Miki K., Cho-Chung Y. S. An antisense oligodeoxynucleotide that depletes RIa subunit of cyclic AMP-dependent protein kinase induces growth inhibition in human cancer cells. Cancer Res. 1993; 53:868-872 [Abstract/Free Full Text]

0022-3166/97 $3.00 ©1997 American Society for Nutritional Sciences