American ginseng transcriptionally activates p21 mRNA in breast cancer cell lines.

American ginseng (AG) has been demonstrated to inhibit breast cancer cell growth in vitro. p21 protein, a universal cell cycle inhibitor, binds cyclin-CDK complexes, an important mechanism in cell cycle regulation. The purpose of this investigation was to determine if AG induces p21 gene expression in hormone sensitive (MCF-7) and insensitive (MDA-MB-231) breast cancer cell lines. Cells grown in steroid stripped medium (SSM) were treated with AG, 17-beta-estradiol (E2), genistein or cycloheximide (CHX). Northern blot analyses were performed using human p21Cip1 and 36B4 cDNA probes. Cell lines were transiently transfected with select mouse p21 CAT reporter constructs, including those lacking a p53 binding site. Cell cycle analyses was performed by FACScan. The results revealed that AG induced p21 mRNA expression in MCF-7 and MDA-MB-231 cells (p=0.0004; p < or =0.0001, respectively). Neither E2 nor genistein alter p21 mRNA expression. CHX, a protein synthesis inhibitor, did not block p21 mRNA expression induced by AG, indicating that p21 is induced as an immediate early gene. AG activated p21 reporter constructs in transfected cells, independent of p53 binding sites. The cell cycle proliferative phase was significantly decreased by AG and increased by E2 (p < or =0.0001). AG may inhibit breast cancer cell growth by transcriptional activation of the p21 gene, independent of p53.

p21 gene regulation during enterocyte differentiation.

BACKGROUND: Enterocyte differentiation is associated with a withdrawal from the cell cycle and the transcriptional activation of the cell cycle inhibitor, p21. We sought to define the molecular mechanisms involved in p21 gene activation in an in vitro system. METHODS: Transient transfections were performed in HT-29 cells with plasmids containing various 5' deletions of the p21 promoter upstream of the luciferase reporter -/+ the histone deacetylase 1 (HDAC1) expression plasmid. After 24 h, cells were treated -/+ 5 mM sodium butyrate (NaBu) or another histone hyperacetylating agent, trichostatin A (TSA, 0.3 microM) for 24 h. After protein extraction, luciferase activity was measured. Acid/urea/triton gel electrophoresis was performed to examine histone acetylation in cells. RESULTS: NaBu and TSA both caused histone H4 hyperacetylation. Both NaBu and TSA caused a marked increase in the transactivation of plasmids containing 291 bp of the p21 promoter upstream of the transcriptional start site, similar to that previously seen for a 2.4-kb construct. A decrease in reporter gene induction was seen between 173 and 153 bp. This was followed by a marked increase in promoter induction from 143 to 117 bp. Finally, only low basal activity was seen in the case of the 93-bp plasmid. HDAC1 blocked NaBu-mediated induction of all plasmids. CONCLUSIONS: p21 gene activation during HT-29 cell differentiation occurs via at least two regions of cis-acting elements: one located between -93 and -117 bp, and the other between -173 and -291 bp. Histone hyperacetylation likely plays a role in this activation.

Transient vs. prolonged histone hyperacetylation: effects on colon cancer cell growth, differentiation, and apoptosis.

The role of histone hyperacetylation in regard to growth, differentiation, and apoptosis in colon cancer cells was assessed in an in vitro model system. HT-29 cells were grown in +/-10% fetal bovine serum with either 5 mM sodium butyrate or 0.3 microM trichostatin A [single dose (T) or 3 doses 8 h apart (TR)] for 24 h. Serum-starved HT-29 cells were further treated with epidermal growth factor or insulin-like growth factor I for an additional 24 h. Apoptosis was quantified with propidium iodide and characterized by electron microscopy. Northern blot analyses were performed with cDNA probes specific for intestinal alkaline phosphatase, Na-K-2Cl cotransporter, the cell cycle inhibitor p21, and the actin control. Flow cytometric analysis revealed a time-dependent growth suppression along with early induction of p21 mRNA in the butyrate, T, and TR groups. Histone hyperacetylation, assessed by acid-urea-triton gel electrophoresis, was transient in the T group but persisted for up to 24 h in the butyrate and TR groups. Induction of apoptosis, growth factor unresponsiveness, and differentiation occurred in the butyrate- and TR-treated cells but not those treated with a single dose of trichostatin A. Thus transient hyperacetylation of histones is sufficient to induce p21 expression and produce cellular growth arrest, but prolonged histone hyperacetylation is required for induction of the programs of differentiation, apoptosis, and growth factor unresponsiveness.

Short-chain fatty acids and thyroid hormone interact in regulating enterocyte gene transcription.

BACKGROUND: Enterocyte differentiation is known to be regulated by a variety of extracellular compounds, among which are triiodothyronine (T3) and the short-chain fatty acids (SCFAs). Because several SCFAs are known to induce histone hyperacetylation, and T3 action has been recently linked to chromatin structure, we sought to investigate the interplay between SCFAs and T3 in regard to the enterocyte differentiation marker, intestinal alkaline phosphatase (IAP). METHODS: Caco-2 cells were transiently transfected with a reporter construct containing 2.4 kb of the human IAP gene 5' flanking region (IAP2.4CAT). Cotransfections were carried out with and without thyroid hormone receptor-1 (TR beta-1) or histone deacetylase-1 (HDAC-1) expression plasmids. Cells were treated with 5 mmol/L SCFAs (propionic, butyric, valeric, or caproic acids as propionate, butyrate, valerate, or caproate, respectively), with and without 10 nmol/L T3. Reporter gene activity was measured and the level of histone acetylation assessed by means of acid-urea-triton (AUT) gel assays. RESULTS: TR beta-1 cotransfection caused a marked decrease in IAP reporter gene activity, which is consistent with the well-known phenomenon of ligand independent repression (LIR), whereas T3 treatment reversed the LIR and caused further reporter gene activation. Treatment with SCFAs similarly resulted in a complete blockage of LIR, and, in fact, turned the TR beta-1 into a transcriptional activator, even in the absence of T3. Concomitant treatment with T3 and butyric acid produced an additive effect on IAP transactivation. In contrast, cotransfection with HDAC-1 attenuated the effects of SCFAs on IAP gene activation. AUT gel studies demonstrated histone hyperacetylation in response to SCFA treatment. CONCLUSION: One or more DNA cis-elements in the human IAP gene mediate ligand independent repression by the TR beta-1, an effect that can be entirely reversed by those SCFAs that induce histone hyperacetylation. In addition T3 and SCFAs can act in concert to induce IAP gene transcription, demonstrating an important link between triiodothyronine and histone hyperacetylation in regard to enterocyte-specific gene expression.

Histone acetylation and cancer.

In the past year, several papers have been published which implicate a link between alterations in chromatin structure and the development of cancer. Both histone hyperacetylation and hypoacetylation appear to be important in the neoplastic process, depending on the target gene involved. In the case of colon cancer, induction of the p21 gene by histone hyperacetylation may be a mechanism by which dietary fiber prevents carcinogenesis.

p21(WAF1) is required for butyrate-mediated growth inhibition of human colon cancer cells.

A diet high in fiber is associated with a decreased incidence and growth of colon cancers. Butyrate, a four-carbon short-chain fatty acid product of fiber fermentation within the colon, appears to mediate these salutary effects. We sought to determine the molecular mechanism by which butyrate mediates growth inhibition of colonic cancer cells and thereby to elucidate the molecular link between a high-fiber diet and the arrest of colon carcinogenesis. We show that concomitant with growth arrest, butyrate induces p21 mRNA expression in an immediate-early fashion, through transactivation of a promoter cis-element(s) located within 1.4 kb of the transcriptional start site, independent of p53 binding. Studies using the specific histone hyperacetylating agent, trichostatin A, and histone deacetylase 1 indicate that growth arrest and p21 induction occur through a mechanism involving histone hyperacetylation. We show the critical importance of p21 in butyrate-mediated growth arrest by first confirming that stable overexpression of the p21 gene is able to cause growth arrest in the human colon carcinoma cell line, HT-29. Furthermore, using p21-deleted HCT116 human colon carcinoma cells, we provide convincing evidence that p21 is required for growth arrest to occur in response to histone hyperacetylation, but not for serum starvation nor postconfluent growth. Thus, p21 appears to be a critical effector of butyrate-induced growth arrest in colonic cancer cells, and may be an important molecular link between a high-fiber diet and the prevention of colon carcinogenesis.

Na-K-2Cl cotransporter gene expression and function during enterocyte differentiation. Modulation of Cl- secretory capacity by butyrate.

The basolateral Na-K-2Cl cotransporter (NKCC1) is a key component of the intestinal crypt cell secretory apparatus. Its fate during the transition to absorptive enterocyte and the potential impact of its altered expression on secretory output have not been addressed. In this report, NKCC1 mRNA was found to be expressed in rat jejunal crypt but not villus cells. Butyrate treatment of intestinal epithelial HT29 cells induced a differentiation pattern that recapitulated the rat intestinal crypt-villus axis, with NKCC1 mRNA levels decreasing in a time- and dose-dependent fashion in parallel with upregulation of apical brush-border markers. Butyrate but not acetate or proprionate decreased basal and cAMP-stimulated bumetanide-sensitive K+ (86Rb) uptake in both HT29 cells and the Cl--secreting T84 line. Butyrate markedly decreased transepithelial Cl- secretion in confluent T84 monolayers without effect on cAMP-regulated apical Cl- efflux. We conclude that NKCC1 regulation during enterocyte differentiation occurs at the level of gene expression, and that selective downregulation of NKCC1 gene expression and function by butyrate leads to a profound decrease in transepithelial Cl- secretion. These data emphasize the importance of NKCC1 in determining epithelial secretory capacity and suggest the possibility of modulation of the enterocytic transport phenotype as therapy for diarrheal disorders.

Periodontal ligament cells and gingival fibroblasts respond differently to attachment factors in vitro.

One of the initial events required for regeneration of periodontal tissues lost due to disease is the establishment of connective tissue attachment to root surfaces. Thus, considerable research efforts have focused on developing reliable procedures to gain new connective tissue attachment. Our studies focus on evaluating agents for their ability to promote cell attachment and spreading using an in vitro assay. For these studies human gingival fibroblasts (GF) and human periodontal ligament (PDL) cells, after exposure to fibronectin; 44 kilodalton bone phosphoprotein (44K BPP-osteopontin) or guanidine EDTA extracts of bone, cementum, or dentin, were compared as to degree of cell attachment and spreading. Fibronectin equally enhanced attachment and spreading PDL cells and GF. In contrast, 44K BPP, as well as guanidine EDTA extracts of bone and cementum, preferentially promoted attachment of GF when compared with attachment of PDL cells. For both PDL cells and GF the attached cells exhibited spreading. The guanidine EDTA extract of dentin did not promote attachment of either cell type. These results suggest that PDL cells and GF have different attachment properties which need to be considered for investigations directed at developing regenerative periodontal treatments.

A comparative study of human periodontal ligament cells and gingival fibroblasts in vitro.

Both periodontal ligament and gingival tissue are thought to harbor cells with the ability to stimulate periodontal regeneration, i.e., formation of new bone, cementum, and connective tissue attachment. To understand further the role of these cells in the regenerative process, we compared human periodontal ligament cells and gingival fibroblasts, both derived from the same patient, same passage, in vitro. Protein and collagen production was significantly greater in periodontal ligament cells when compared with that of gingival fibroblasts. In addition, periodontal ligament cells had higher alkaline phosphatase levels when compared with those of gingival fibroblasts.

Enhancement by extracts of mineralized tissues of protein production by human gingival fibroblasts in vitro.

Non-confluent cell cultures were exposed to both guanidine and guanidine-EDTA extracts of cementum, dentine and alveolar bone, at concentrations from 2 to 50 micrograms/ml for 48 h. The cells were radioactively labelled during the last 24 h. Total protein production was measured via incorporation of radioactive proline; collagen production was estimated by digestion of the radioactive protein mixture with bacterial collagenase. All guanidine-EDTA extracts elicited statistically-significant increases in total protein production when compared to controls. At 50 micrograms/ml of extract, the increase in protein production was 340, 143 and 338 per cent for bone, cementum and dentine, respectively. Similar results were obtained for collagen production. Guanidine-EDTA extracts also stimulated an increase in the production of specific proteins, as ascertained by gel electrophoresis. In contrast, the guanidine extracts had no effect on either protein or collagen production. Thus the functions of gingival fibroblasts can be altered by proteins from associated mineralized tissues. Identification of such proteins and their biological functions would enhance knowledge of the mechanisms that regulate connective-tissue regeneration.

Effects of head position on intraoral pressures in Class I and Class II adults.

Variations in resting lip and tongue pressures and their relationship to alterations in head posture were investigated in subjects with Class I and Class II dental and skeletal morphology. Intraoral pressures were measured by means of transducers mounted in plastic carriers, customized for each subject from dental casts. Transducers were placed in the carriers to measure posterior lingual, anterior lingual, and labial pressures along the mandibular dentition. Ten Class I subjects and eleven Class II subjects participated. Pressures were recorded in natural head position, with 20 degrees of head extension and 20 degrees of head flexion. The Friedman two-way analysis of variance using ranked data was used to compare transducer location and head posture within skeletal classes. Anterior pressures were found to differ from posterior pressures in both classes. In Class I subjects, posterior lingual pressures were consistently different from labial pressures in all head positions. In Class II subjects, posterior lingual pressures differed from labial pressures in flexion and natural head positions, and from anterior lingual pressures in flexion and natural head positions. No increase in labial pressures with head extension was found in either Class I or Class II samples. Since every subject showed pressure changes with changes in head position, the influence of posture should be considered in studies on facial morphology and dental equilibrium.

Lip morphology and area changes associated with surgical correction of mandibular prognathism.

Changes in lip morphology and area, measured in two dimensions from standardized lateral head films, were assessed in a series of twenty adults at three times: pre-surgically, 8--14 months post-surgically, and a long-term follow-up at 5--7 years. All individuals received the same, single surgical procedure (Obwegeser sagittal split for correcting mandibular prognathism. Upper and lower lip changes were quantified as millimetres displacement of the lip centroid vertically and horizontally, plus changes in cross-sectional area. Direction and amount of change, its dependency on the amount and kind of surgically induced symphyseal changes, and the intercorrelations among lips and among lip and symphysis variables were statistically evaluated, both univariately and multivariately. Three measures are made of symphysis change: horizontal and vertical repositioning and amount and direction of rotation. Horizontal repositioning primarily affected the lengthening and areal increase of the upper lip. Vertical repositioning had its major influence in the height and cross-sectional area of the lower lip: a superior shift of the mandible made the lower lip shorter, more protrusive and smaller in area; an inferior shift produced an increase in lower lip height with increased area. The third variable, symphyseal rotation, had its greatest influence on the labial-lingual shift of the upper lip's centroid. The long-term follow-up showed little change from the 1 year post-operative conditions; equilibrium of the soft tissue components was then achieved fairly soon after surgery.