p21WAF1 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.

Inhibition of advanced glycation endproduct formation by acetaldehyde: Role in the cardioprotective effect of ethanol

Epidemiological studies suggest that there is a beneficial effect of moderate ethanol consumption on the incidence of cardiovascular disease. Ethanol is metabolized to acetaldehyde, a two-carbon carbonyl compound that can react with nucleophiles to form covalent addition products. We have identified a biochemical modification produced by the reaction of acetaldehyde with protein-bound Amadori products. Amadori products typically arise from the nonenzymatic addition of reducing sugars (such as glucose) to protein amino groups and are the precursors to irreversibly bound, crosslinking moieties called advanced glycation endproducts, or AGEs. AGEs accumulate over time on plasma lipoproteins and vascular wall components and play an important role in the development of diabetes- and age-related cardiovascular disease. The attachment of acetaldehyde to a model Amadori product produces a chemically stabilized complex that cannot rearrange and progress to AGE formation. We tested the role of this reaction in preventing AGE formation in vivo by administering ethanol to diabetic rats, which normally exhibit increased AGE formation and high circulating levels of the hemoglobin Amadori product, HbA1c, and the hemoglobin AGE product, Hb-AGE. In this model study, diabetic rats fed an ethanol diet for 4 weeks showed a 52% decrease in Hb-AGE when compared with diabetic controls (P < 0.001). Circulating levels of HbA1c were unaffected by ethanol, pointing to the specificity of the acetaldehyde reaction for the post-Amadori, advanced glycation process. These data suggest a possible mechanism for the so-called “French paradox,” (the cardioprotection conferred by moderate ethanol ingestion) and may offer new strategies for inhibiting advanced glycation.

Modification of Carbon Partitioning, Photosynthetic Capacity, and O2 Sensitivity in Arabidopsis Plants with Low ADP-Glucose Pyrophosphorylase Activity1

Wild-type Arabidopsis plants, the starch-deficient mutant TL46, and the near-starchless mutant TL25 were evaluated by noninvasive in situ methods for their capacity for net CO2 assimilation, true rates of photosynthetic O2 evolution (determined from chlorophyll fluorescence measurements of photosystem II), partitioning of photosynthate into sucrose and starch, and plant growth. Compared with wild-type plants, the starch mutants showed reduced photosynthetic capacity, with the largest reduction occurring in mutant TL25 subjected to high light and increased CO2 partial pressure. The extent of stimulation of CO2 assimilation by increasing CO2 or by reducing O2 partial pressure was significantly less for the starch mutants than for wild-type plants. Under high light and moderate to high levels of CO2, the rates of CO2 assimilation and O2 evolution and the percentage inhibition of photosynthesis by low O2 were higher for the wild type than for the mutants. The relative rates of 14CO2 incorporation into starch under high light and high CO2 followed the patterns of photosynthetic capacity, with TL46 showing 31% to 40% of the starch-labeling rates of the wild type and TL25 showing less than 14% incorporation. Overall, there were significant correlations between the rates of starch synthesis and CO2 assimilation and between the rates of starch synthesis and cumulative leaf area. These results indicate that leaf starch plays an important role as a transient reserve, the synthesis of which can ameliorate any potential reduction in photosynthesis caused by feedback regulation.

High-Temperature Perturbation of Starch Synthesis Is Attributable to Inhibition of ADP-Glucose Pyrophosphorylase by Decreased Levels of Glycerate-3-Phosphate in Growing Potato Tubers1

To investigate the short-term effect of elevated temperatures on carbon metabolism in growing potato (Solanum tuberosum L.) tubers, developing tubers were exposed to a range of temperatures between 19°C and 37°C. Incorporation of [14C]glucose (Glc) into starch showed a temperature optimum at 25°C. Increasing the temperature from 23°C or 25°C up to 37°C led to decreased labeling of starch, increased labeling of sucrose (Suc) and intermediates of the respiratory pathway, and increased respiration rates. At elevated temperatures, hexose-phosphate levels were increased, whereas the levels of glycerate-3-phosphate (3PGA) and phosphoenolpyruvate were decreased. There was an increase in pyruvate and malate, and a decrease in isocitrate. The amount of adenine diphosphoglucose (ADPGlc) decreased when tubers were exposed to elevated temperatures. There was a strong correlation between the in vivo levels of 3PGA and ADPGlc in tubers incubated at different temperatures, and the decrease in ADPGlc correlated very well with the decrease in the labeling of starch. In tubers incubated at temperatures above 30°C, the overall activities of Suc synthase and ADPGlc pyrophosphorylase declined slightly, whereas soluble starch synthase and pyruvate kinase remained unchanged. Elevated temperatures led to an activation of Suc phosphate synthase involving a change in its kinetic properties. There was a strong correlation between Suc phosphate synthase activation and the in vivo level of Glc-6-phosphate. It is proposed that elevated temperatures lead to increased rates of respiration, and the resulting decline of 3PGA then inhibits ADPGlc pyrophosphorylase and starch synthesis.