Effects of Butyrate on Cell Cycle Progression and Polyploidization of Various Types of Mammalian Cells

We studied the effect of butyrate on cell cycle progression and polyploidization in three fibroblast (rat 3Yl, human IMR-90, and human embryo lung HEL) and two epithelial (human embryo kidney HEK and monkey kidney BSC-1) cells. In these cells, except for 3Y1, G1 arrest with butyrate was incomplete, and the production of tetraploid cells was detectable in the presence of butyrate. G2 arrest with butyrate was also incomplete in HEL and BSC-1 cells, and the number of HEL cells increased in the presence of butyrate. On the contrary, most BSC-1 cells that divided in the presence of butyrate were unstable and the number of attached cells decreased. These results indicate that the effect of butyrate on cell cycle progression varies with the cell type and that polyploidization can be induced by a single treatment with butyrate.     

Oncoprotein 18 levels and phosphorylation mediate megakaryocyte polyploidization in human erythroleukemia cells

Megakaryocytes undergo an unusual cell cycle during differentiation that results in polyploidy through largely unknown mechanism(s). It has been shown that serine phosphorylation of oncoprotein 18 (Op18) is required for cell cycle progression specifically at the G2/M transition. Moreover, mutant forms of Op18 that are defective in one or more of the four serine residues induce G2/M arrest and subsequent polyploidization. Op18 phosphorylation is rapidly induced with phorbol myristate acetate (PMA) treatment in a wide range of human cells. In this study, we investigated the role of Op18 in PMA induced polyploidization during megakaryocyte differentiation of the human erythroleukemia cell line. Crucial to the molecular analysis of megakaryocyte differentiation, is the ability to fractionate cell populations with different ploidy levels. We have utilized cell elutriation as a fractionation strategy to analyze Op18 expression in synchronized cell subpopulations in different phases of the cell cycle or with progressive megakaryocyte polyploidization. In the absence of PMA, increased phosphorylation of Op18 was observed in HEL cells during cell cycle progression, as for other proliferating cells. However, in contrast to Jurkat leukemia cells chosen as control, HEL cells exhibited a lack of Op18 phosphorylation in response to PMA, which was accompanied by polyploidization and differentiation along the megakaryocytic lineage. To further determine the role of Op18 in polyploidization, HEL cells were transfected with different Op18 expression constructs. Differences in cell survival and polyploidization were observed between high and low Op18 expressors. An increased Op18 level reduced cell survival during the early stage of PMA induced megakaryocyte differentiation, but enhanced polyploidization efficiency. Our findings suggest that maintenance of a high level of unphosphorylated Op18 is required for efficient polyploidization during the differentiation program of megakaryocytes.     

Induction of premature senescence program by an inhibitor of histone deacetylase sodium butyrate in normal and transformed rat fibroblasts

We investigated a possibility to induce the premature cell senescence in rat embryo fibroblasts and E1A + cHa-ras transformants. We found that after the treatment with sodium butyrate, an inhibitor of histone deacetylases, both normal and transformed cells completely stopped to proliferate and accumulated at G1/S and G2/M phases of the cell cycle. The cloning efficiency data show that the cell cycle arrest induced by sodium butyrate is irreversible and correlates with the accumulation of active phosphorylated form of stress kinase p38, and with the expression of marker of senescence--beta-galactosidase activity (SA beta-Gal). The program resembling the premature senescence after sodium butyrate treatment is supposed to develop both in normal and transformed cells. The irreversible block of proliferation in E1A + cHa-ras transformants may be regarded as an example of activation of anticancer program like that of premature senescence in the tumor rodent cells.     

Sodium butyrate induces G2 arrest in the human breast cancer cells MDA-MB-231 and renders them competent for DNA rereplication

When exposed to sodium butyrate (NaBut), exponentially growing cells accumulate in G1 and G2 phases of the cell cycle. In the human breast cancer cell line MDA-MB-231, an arrest in G2 phase was observed when the cells were released from hydroxyurea block (G1/S interface) in the presence of NaBut. The inhibition of G2 progression was correlated with increased contents both of total p21(Waf1) and of p21(Waf1) associated with cyclin A and with an inhibition of cyclin A- and B1-associated histone H1 kinase activities measured in cell lysates, as well as with dephosphorylation of the RB protein. A decrease in the cell contents of cyclins A and B1 was also observed but this decrease was preceded by p21(Waf1) accumulation. When NaBut was removed from the culture medium of cells blocked in G2 phase, p21(Waf1) level decreased and, instead of proceeding to mitosis, these cells resumed a progression toward DNA rereplication. These results suggest that the induction of p21(Waf1) by NaBut leads to the inhibition of the sequential activation of cyclin A- and B1-dependent kinases in this cell line, resulting in the inhibition of G2 progression and rendering the cells competent for a new cell division cycle.     

Hypophosphorylation of the RB Protein in S and G2 as Well as G1 during Growth Arrest

The RB tumor suppressor protein is a cell cycle regulator, where hypophosphorylated RB is associated with G1/0 arrest and its cyclin-dependent phosphorylation in G1 allows progression from G1 to S. The present report shows that in human leukemia cells induced to undergo growth arrest with sodium butyrate or DMSO, hypophosphorylation of the RB protein is not G1 restricted and also occurs in S and G2/M cells as well as in G1 cells when growth is inhibited. While all of the RB protein in G1/0 cells is hypophosphorylated, residual cells in S and G2 have significant detectable amounts of hypophosphorylated RB as well as still hyperphosphorylated RB protein. Thus RB hypophosphorylation can be induced in S and G2 as well as the G1 phase. The results show that growth retardation in other than the G1 phase is associated with occurrence of hypophosphorylated RB. RB may thus have a broader capability to inhibit proliferation than just in G1.     

Expression of c-fos oncogene during hepatocarcinogenesis, liver regeneration and in synchronized HTC cells

We have studied the expression of c-fos gene in rat hepatoma induced by DENA. An increase of c-fos mRNA concentration was observed after 8 days, but the maximal 5- to 6-fold increase was observed after 70 weeks. This increase was found in perinodular hepatocytes as well as in cancer nodules. c-fos expression was also enhanced during liver regeneration at a period corresponding to cell proliferation. In HTC cells the arrest of the cell cycle at early G1 phase by addition of sodium butyrate was accompanied by a strong increase of c-fos gene expression. However the c-fos mRNA rapidly decreased after removal of sodium butyrate during the progression of the cells in the cell cycle and increased transiently when the cells entered again in G1 phase.     

Extent of histone modifications and H1 content during cell cycle progression in the presence of butyrate

The effects of butyrate upon the extents of phosphorylation of histones H1 and H1(0) during cell-cycle progression have been investigated. Chinese hamster (line CHO) cells were synchronized in early S phase and released into medium containing 0 or 15 mM butyrate to resume cell-cycle traverse into G1 of the next cell cycle. Cells were also mechanically selected from monolayer cultures grown in the presence of colcemid and 0 or 15 mM butyrate to obtain greater than 98% pure populations of metaphase cells. Although cell cycle progression is altered by butyrate, electrophoretic patterns of histones H1, H1(0), H3, and H4 indicate that butyrate has little, if any, effect on the extents of H1 and H1(0) phosphorylation during the cell cycle or the mitotic-specific phosphorylation of histone H3. Butyrate does, however, inhibit removal of extraordinary levels of histone H4 acetylation (hyperacetylation) during metaphase, and it appears to cause an increase in the content of H1(0) in chromatin during the S or G2 phases of the cell cycle.     

Influence of adeno-associated virus on adherence and growth properties of normal cells.

It has been shown previously that infection of newly established cell cultures from malignant human tumors with adeno-associated parvovirus type 2 or type 5 results in growth arrest and cell death. Here we report that the additionally observed antiproliferative effect on diploid human fibroblasts is transient and is connected to a reduced number of cells in S phase. Progression through the cell cycle is disturbed either in G0/G1 or at the G1/S boundary, but an additional arrest in G2 cannot be excluded. DNA synthesis and cell proliferation are resumed when cells are recultured after loosening of cell-matrix adhesions by trypsin treatment. In contrast, they are not resumed by solely providing growth factors via higher amounts of fetal calf serum. The results suggest that cell adherence is altered in adeno-associated parvovirus-infected human embryo fibroblasts.     

Novel response to microtubule perturbation in meiosis

During the mitotic cell cycle, microtubule depolymerization leads to a cell cycle arrest in metaphase, due to activation of the spindle checkpoint. Here, we show that under microtubule-destabilizing conditions, such as low temperature or the presence of the spindle-depolymerizing drug benomyl, meiotic budding yeast cells arrest in G(1) or G(2), instead of metaphase. Cells arrest in G(1) if microtubule perturbation occurs as they enter the meiotic cell cycle and in G(2) if cells are already undergoing premeiotic S phase. Concomitantly, cells down-regulate genes required for cell cycle progression, meiotic differentiation, and spore formation in a highly coordinated manner. Decreased expression of these genes is likely to be responsible for halting both cell cycle progression and meiotic development. Our results point towards the existence of a novel surveillance mechanism of microtubule integrity that may be particularly important during specialized cell cycles when coordination of cell cycle progression with a developmental program is necessary.     

EGF induces cell cycle arrest of A431 human epidermoid carcinoma cells

The human carcinoma cell line A431 is unusual in that physiologic concentrations of epidermal growth factor (EGF) inhibit proliferation. In the presence of 5-10 nM EGF proliferation of A431 cells is abruptly and markedly decreased compared to the untreated control cultures, with little loss of cell viability over a 4-day period. This study was initiated to examine how EGF affects the progression of A431 cells through the cell cycle. Flow cytometric analysis of DNA in EGF-treated cells reveals a marked change in the cell cycle distribution. The percentage of cells in late S/G2 increases and early S phase is nearly depleted. Since addition of the mitotic inhibitor vinblastine causes accumulation of cells in mitosis and prevents reentry of cells into G1, it is possible to distinguish between slow progression through G1 and G2 and blocks in those phases. When control cells, not treated with EGF, are exposed to vinblastine, the cells accumulate mitotic figures, as expected, and show progression into S, thus diminishing the number of cells in G1. In contrast, no mitotic figures are found among the EGF-treated cells in the presence or absence of vinblastine, and progression from G1 into S is not observed, as the number of cells in G1 remains constant. These results suggest that there are two EGF-induced blocks in cell cycle transversal; one is in late S and/or G2, blocking entry into mitosis, and the other is in G1, blocking entry into S phase. After 24 hours of EGF treatment, DNA synthesis is reduced to less than 10% compared to untreated controls as measured by the incorporation of [3H]thymidine or BrdU. In contrast, protein synthesis is inhibited by about twofold. Although inhibition of protein synthesis is less extensive, it occurs 6 hours prior to an equivalent inhibition of DNA synthesis. The rapid decrease in protein synthesis may result in the subsequent cell cycle arrest which occurs several hours later.     

G2 arrest, binucleation, and single-parameter DNA flow cytometric analysis

One important facet of flow cytometry involves the effects of pharmacological agents on cell cycle progression. Comparative G2 fraction perturbations were examined: effects of sodium butyrate on articular chondrocytes, effects of an antineoplastic agent (SOAZ) and an antirheumatic drug (D-penicillamine) on HeLa cells. Even though DNA flow cytometric analysis detects preferentially an induction of G2 arrest, the mode of action of these agents on the cell cycle is different. Sodium butyrate and D-penicillamine lead to an increase of binucleate cells due to cytokinesis perturbation. Because of similar fluorescence intensity, distinguishing G2 from binucleate GO/1 cells is not easily possible using DNA content measurement and reflects a failure of flow cytometry in the detection of binucleate cells. Rapid cell cycle analysis of single cells should contribute greatly to the study of pharmacological interactions, but DNA flow cytometric measurements obtained from cultured cells exposed to certain agents must be cautiously interpreted because those may interact on cytokinesis and induce artefacts in histogram interpretation.     

Differentiation of HL-60 myeloid leukaemia cells is associated with a transient block in the G2 phase of the cell cycle

The human promyelocytic leukaemia cell line HL-60 can be induced to differentiate towards mature granulocytes by treatment with dibutyryl cyclic adenosine-3',5'-monophosphate (dbcAMP). Differentiation begins within 16-24 h of treatment and is associated with a time- and dose-dependent accumulation of cells in the G0/G1 phase of the cell cycle with a concomitant decrease in the number of cells in the S and G2 + M phases. Using acridine orange staining, we found that the RNA content of the cells also decreased following differentiation. Stathmokinetic analysis of HL-60 cell populations following dbcAMP treatment showed no effect on the total number of cells in the G0/G1 or S phases, or the rate of progression of cells through these cell cycle compartments. In contrast, dbcAMP was found to induce a transient arrest of the cells in the G2 phase. We also found that differentiation induced by dbcAMP did not require progression of the cells through the cell cycle. Cells arrested in either G1/S by hydroxyurea or G2 + M by colcemid eventually expressed markers of mature granulocytes. These results demonstrate that dbcAMP modulates cell cycle progression. However, these cell cycle changes alone are insufficient to induce granulocytic differentiation of HL-60 cells.     

Butyrate-induced apoptotic cascade in colonic carcinoma cells: modulation of the beta-catenin-Tcf pathway and concordance with effects of sulindac and trichostatin A but not curcumin.

Short-chain fatty acids play a critical role in colonic homeostasis because they stimulate pathways of growth arrest, differentiation, and apoptosis. These effects have been well characterized in colonic cell lines in vitro. We investigated the role of beta-catenin-Tcf signaling in these responses to butyrate and other well-characterized inducers of apoptosis of colonic epithelial cells. Unlike wild-type APC, which down-regulates Tcf activity, butyrate, as well as sulindac and trichostatin A, all inducers of G0-G1 cell cycle arrest and apoptosis in the SW620 colonic carcinoma cell line, up-regulate Tcf activity. In contrast, structural analogues of butyrate that do not induce cell cycle arrest or apoptosis and curcumin, which stimulates G2-M arrest without inducing apoptosis, do not alter Tcf activity. Similar to the cell cycle arrest and apoptotic cascade induced by butyrate, the up-regulation of Tcf activity is dependent upon the presence of a mitochondrial membrane potential, unlike the APC-induced down-regulation, which is insensitive to collapse of the mitochondrial membrane potential. Moreover, the butyrate-induced increase in Tcf activity, which is reflected in an increase in beta-catenin-Tcf complex formation, is independent of the down-regulation caused by expression of wild-type APC. Thus, butyrate and wild-type APC have different and independent effects on beta-catenin-Tcf signaling. These data are consistent with other reports that suggest that the absence of wild-type APC, associated with the up-regulation of this signaling pathway, is linked to the probability of a colonic epithelial cell entering an apoptotic cascade.     

Effect of sodium butyrate on cell proliferation and cell cycle in porcine intestinal epithelial (IPEC-J2) cells

Conflicting results have been reported that butyrate in normal piglets leads either to an increase or to a decrease of jejunal villus length, implying a possible effect on the proliferation of enterocytes. No definitive study was found for the biological effects of butyrate in porcine jejunal epithelial cells. The present study used IPEC-J2 cells, a non-transformed jejunal epithelial line to evaluate the direct effects of sodium butyrate on cell proliferation, cell cycle regulation, and apoptosis. Low concentrations (0.5 and 1 mM) of butyrate had no effect on cell proliferation. However, at 5 and 10 mM, sodium butyrate significantly decreased cell viability, accompanied by reduced levels of p-mTOR and PCNA protein. Sodium butyrate, in a dose-dependent manner, induced cell cycle arrest in G0/G1 phase and reduced the numbers of cells in S phase. In addition, relative expression of p21, p27, and pro-apoptosis bak genes, and protein levels of p21Waf1/Cip1, p27Kip1, cyclinD3, CDK4, and Cleave-caspase3 were increased by higher concentrations of sodium butyrate (1, 5, 10 mM), and the levels of cyclinD1 and CDK6 were reduced by 5 and 10 mM butyrate. Butyrate increased the phosphorylated form of the signaling molecule p38 and phosphorylated JNK. In conclusion, the present in vitro study indicated that sodium butyrate inhibited the proliferation of IPEC-J2 cells by inducing cell cycle arrest in the G0/G1 phase of cell cycles and by increasing apoptosis at high concentrations.     

Alpha-lipoic acid induces p27Kip-dependent cell cycle arrest in non-transformed cell lines and apoptosis in tumor cell lines

alpha-Lipoic acid is a naturally-occurring co-factor found in a number of multi-enzyme complexes regulating metabolism. We report here that alpha-lipoic acid induces hyperacetylation of histones in vivo and has differential effects on the growth and viability of normal versus transformed cell lines. The human tumor cell lines FaDu and Jurkat, as well as a Ki-v-Ras-transformed Balb/c-3T3 murine mesenchymal cell line, all initiated apoptosis following exposure to alpha-lipoic acid. In contrast, treatment of non-transformed cell lines with alpha-lipoic acid resulted only in reversible cell cycle arrest in G0/G1. Treatment with butyrate, another short-chain fatty acid, induced a G0/G1 arrest in both transformed and non-transformed cell lines. alpha-Lipoic acid caused a post-translational elevation in the levels of the cyclin-dependent kinase inhibitor p27Kip1. Studies using p27Kip1-deficient MEF cells demonstrated that p27Kip1 was required for the alpha-lipoic acid-mediated cell cycle arrest. The mechanism of apoptosis was independent of Fas-mediated signaling, as alpha-lipoic acid-treated Jurkat cell mutants deficient in Fas or FADD retained sensitivity to apoptosis. The differential selectivity of the pro-apoptotic effects of alpha-lipoic acid for transformed cells supports its potential use in the treatment of neoplastic disorders.     

Enhanced sensitivity of G1 arrested human cancer cells suggests a novel therapeutic strategy using a combination of simvastatin and TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells over normal cells. To study the relationship between cell cycle progression and TRAIL-induced apoptosis, SW480 colon cancer and H460 lung cancer cell lines were examined for their sensitivity to TRAIL after arrest in different cell cycle phases. Cells were synchronized in G0/G1, S, and G2/M phase by serum starvation, aphidicolin, or nocodazole treatment, respectively. We found that arrest of cells in G0/G1 phase confers significantly higher susceptibility to TRAIL-induced apoptosis as compared to cells in late G1, S, or G2/M phase. To determine if cell cycle phase could be harnessed for therapeutic gain in the presence of TRAIL, we used the HMG-CoA reductase inhibitor, Simvastatin and lovastatin, to enrich a cancer cell population in G0/G1. Both simvastatin and lovastatin significantly augmented TRAIL-induced apoptosis in tumor cells, but not in normal keratinocytes. The results indicate that TRAIL, in combination with a HMG-CoA reductase inhibitor, may have therapeutic potential in the treatment of human cancer.     

The role of a cell surface inhibitor in early signal transduction associated with the regulation of cell division and differentiation

Serum stimulation of quiescent human fibroblast cultures resulted in a hyperphosphorylation of the nuclear retinoblastoma gene susceptibility product (RB). However, serum stimulation in the presence of 9 x 10(-8) M of a purified bovine sialoglycopeptide (SGP) cell surface inhibitor abrogated the hyperphosphorylation of the RB protein and the subsequent progression of cells through the mitotic cycle. The experimental results suggest that the SGP mediated its cell cycle arrest at a site in the cell cycle that was at the time of RB phosphorylation or somewhat upstream of the modification of this regulatory protein of cell division. Both cells serum-deprived and serum stimulated in the presence of the SGP displayed only a hypophosphorylated RB protein, consistent with the SGP-mediated cell cycle arrest point being near the G1/S interface.