Krüppel-like factor 4 exhibits antiapoptotic activity following gamma-radiation-induced DNA damage.

In response to gamma-radiation-induced DNA damage, organisms either activate cell cycle checkpoint and repair machinery or undergo apoptosis to eliminate damaged cells. Although previous studies indicated that the tumor suppressor p53 is critically involved in mediating both responses, how a cell decides which pathway to take is not well established. The zinc-finger-containing transcription factor, Krüppel-like factor 4 (KLF4), is a crucial mediator for the checkpoint functions of p53 after gamma-irradiation and does so by inhibiting the transition from the G(1) to S and G(2) to M phases of the cell cycle. Here, we determined the role of KLF4 in modulating the apoptotic response following gamma-irradiation. In three independent cell systems including colorectal cancer cells and mouse embryo fibroblasts in which expression of KLF4 could be manipulated, we observed that gamma-irradiated cells underwent apoptosis if KLF4 was absent. In the presence of KLF4, the degree of apoptosis was significantly reduced and cells resorted to checkpoint arrest. The mechanism by which KLF4 accomplished this antiapoptotic effect is by activating expression of the cell cycle arrest gene, p21(WAF1/CIP1), and by inhibiting the ability of p53 to transactivate expression of the proapoptotic gene, BAX. Results of our study illustrate an unexpected antiapoptotic function of KLF4, heretofore considered a tumor suppressor in colorectal cancer, and suggest that KLF4 may be an important determinant of cell fate following gamma-radiation-induced DNA damage.

Foxl1 controls the Wnt/beta-catenin pathway by modulating the expression of proteoglycans in the gut.

Foxl1 is a winged helix transcription factor expressed in the mesenchyme of the gastrointestinal tract. Foxl1 null mice display severe structural defects in the epithelia of the stomach, duodenum, and jejunum. Here we addressed the molecular mechanisms by which Foxl1 controls gastrointestinal differentiation. First we showed that the abnormalities found in the epithelia of the null mice are the result of an increase in the number of proliferating cells and not a change in the rate of cell migration. Next we investigated the regulatory circuits affected by Foxl1. We focused on the Wnt/beta-catenin signaling pathway as a possible target of Foxl1 as it has been shown to play a central role in gastrointestinal proliferation. We demonstrated that Foxl1 activates the Wnt/beta-catenin pathway by increasing extracellular proteoglycans, which act as co-receptors for Wnt. Thus we establish that Foxl1 is involved in the regulation of the Wnt/beta-catenin pathway, providing a novel link in mesenchymal/epithelial cross-talk in the gut. Moreover, we provide the first example implicating proteoglycans in the regulation of cellular proliferation in the gastrointestinal tract.

Intestinal inflammation reduces expression of DRA, a transporter responsible for congenital chloride diarrhea.

The pathogenesis of diarrhea in intestinal inflammatory states is a multifactorial process involving the effects of inflammatory mediators on epithelial transport function. The effect of colonic inflammation on the gene expression of DRA (downregulated in adenoma), a chloride-sulfate anion transporter that is mutated in patients with congenital chloridorrhea, was examined in vivo as well as in an intestinal epithelial cell line. DRA mRNA expression was diminished five- to sevenfold in the HLA-B27/beta2m transgenic rat compared with control. In situ hybridization showed that DRA, which is normally expressed in the upper crypt and surface epithelium of the colon, was dramatically reduced in the surface epithelium of the HLA-B27/beta2m transgenic rat, the interleukin-10 (IL-10) knockout mouse with spontaneous colitis, and in patients with ulcerative colitis. Immunohistochemistry demonstrated that mRNA expression of DRA reflected that of protein expression in vivo. IL-1beta reduced DRA mRNA expression in vitro by inhibiting gene transcription. The loss of transport function in the surface epithelium of the colon by attenuation of transporter gene expression, perhaps inhibited at the level of gene transcription by proinflammatory cytokines, may play a role in the pathogenesis of diarrhea in colitis.

Rheumatologic manifestations of gastrointestinal diseases.

One of the most significant extraintestinal manifestations of gastrointestinal diseases is rheumatologic disorders. The gastrointestinal diseases with rheumatologic manifestations can be divided into two major categories: intestinal disorders and disorders of the liver, biliary tree, and pancreas. The cause of diseases in each of these categories is different. Although intestinal permeability and immune responsiveness are frequently implicated in disorders of the intestine, diseases of the liver, biliary tree, and pancreas usually involve the production of autoantibodies, cytokines, or enzymes. Treatment of rheumatologic complications frequently involves therapy directed at the underlying gastrointestinal disease.

Enhanced external counterpulsation as an adjunct to revascularization in unstable angina.

Enhanced external counterpulsation (EECP) is an effective noninvasive treatment for chronic stable angina. Despite intensive risk factor modification, a patient required two surgical coronary revascularizations and seven multivessel angioplasties over a 26-month period, demonstrating recurrent unstable angina and persistent thallium perfusion defects despite revascularization. Post EECP, angina was relieved, thallium defects were resolved and the patient has remained asymptomatic for 36 months.

Inhibition of IL-8 gene expression in Caco-2 cells by compounds which induce histone hyperacetylation.

Ulcerative colitis, an idiopathic inflammatory disease of the colonic mucosa, can be effectively treated by enemas containing short chain fatty acids (SCFA) such as butyrate, propionate, and acetate. The molecular mechanisms that lead to this response have not been well characterized. It is well known that intestinal inflammation leads to an alteration in patterns of epithelial differentiation with an increase in epithelial proliferation and an expansion of cell populations in an undifferentiated state. SCFAs such as butyrate are capable of inhibiting cell proliferation and inducing a differentiated phenotype in vitro. The Caco-2 colon cancer cell line was used to study the effect of SCFAs and the process of cellular differentiation on the expression of the pro-inflammatory cytokine, interleukin 8 (IL-8). SCFAs and trichostatin A, structurally unrelated compounds which both induce histone hyperacetylation, both led to a dose-dependent inhibition of IL-8 gene expression. Furthermore, spontaneous differentiation of Caco-2 cells by growth to a post-confluent state also inhibited the expression of IL-8. A possible mechanism by which SCFAs may be effective in the treatment of ulcerative colitis may be through their ability to increase histone acetylation states and inhibit the production of pro-inflammatory substances by the intestinal epithelium.

Vitamin K-dependent clotting factors are elevated in young adults who have close relatives with ischemic heart disease.

An increase in factor VII was found to be a risk factor for ischemic heart disease. The present study was designed to test the hypothesis that this increase in factor VII is part of a general increase in vitamin K-dependent clotting factors. Initially, a prospective analysis of factor VII antigen and prothrombin activity was performed in two groups of young subjects without symptoms who differed in their risk of ischemic heart disease based on a history (or lack thereof) of premature heart disease in a first-degree relative. A statistically significant increase in prothrombin activity and factor VII antigen was found in the high-risk group of subjects when compared with the low-risk group. In a second series of subjects, factor IX and X activity assays were also performed, and all four of the vitamin K-dependent clotting factors were found to be significantly higher in high-risk subjects when compared with low-risk subjects. A second goal of the study was to explore whether correlations between factor VII and cholesterol and triglycerides might be due to binding of factor VII with apolipoprotein B. Although a significant correlation of factor VII antigen with apolipoprotein B (rho = 0.523, p < 0.025) was found in our high-risk group of subjects, the correlation between factor VII and triglycerides (rho = 0.641, p < 0.005) was even stronger statistically, suggesting a probable interaction of factor VII with very-low-density lipoproteins in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma fibrinogen level is not elevated in young adults from families with premature ischemic heart disease.

Several epidemiological studies have found that the plasma fibrinogen level is a risk factor for ischemic heart disease (IHD), similar in importance to the serum cholesterol level. A family history of IHD is also a significant risk factor for IHD, statistically independent of the serum cholesterol level. Whether the familial risk for IHD is related to genetic control of the fibrinogen level is unknown. Estimates of the genetic contribution to the variance in plasma fibrinogen levels vary markedly. We previously found elevated levels of cholesterol and factor VII in young subjects with a familial history of premature IHD. In the present study we chose to measure fibrinogen, factor VII antigen, and total cholesterol levels in 43 asymptomatic first-degree relatives (< 50 years old) of patients with premature IHD and in 43 age- and sex-matched asymptomatic young adults at low risk of IHD. No subjects in either group were smokers. The mean plasma fibrinogen level of the high-risk group (259 mg/dL) did not differ significantly from that of the low-risk group (250 mg/dL; p > 0.4). In contrast, the high-risk group had significantly higher mean factor VII antigen (p < 0.001) and mean serum cholesterol (p < 0.0001) than the low-risk group. These data argue against the hypothesis that genetic determination of the plasma fibrinogen level is a common pathophysiological mechanism responsible for familial risk of IHD.

Quantitative polymerase chain reaction analysis of herpes simplex virus DNA in ganglia of mice infected with replication-incompetent mutants.

To study the roles of viral genes in the establishment and maintenance of herpes simplex virus (HSV) latency, we have developed a polymerase chain reaction assay that is both quantitative and sensitive. Using this assay, we analyzed the levels of viral DNA in trigeminal ganglia of mice inoculated corneally with HSV mutants that are defective for virus replication at one or more sites in mice and for reactivation upon ganglionic explant. Ganglia from mice infected with thymidine kinase-negative mutants, which replicate at the site of inoculation and establish latency but do not replicate acutely in ganglia or reactivate upon explant, contained a range of levels of HSV DNA that overlapped with the range found in ganglia latently infected with wild-type virus. On average, these mutant-infected ganglia contained one copy of HSV DNA per 100 cell equivalents (ca. 10(4) molecules), which was 50-fold less than the average for wild-type virus. Ganglia from mice infected with a ribonucleotide reductase deletion mutant, which is defective for acute replication and reactivation upon ganglionic explant, also contained on average one copy of HSV DNA per 100 cell equivalents. We also detected substantial numbers of HSV DNA molecules (up to ca. 10(3] in ganglia of mice infected with an ICP4 deletion mutant and other replication-negative mutants that are severely impaired for viral DNA replication and gene expression. These results raise the possibility that such mutants can establish latency, which could have important implications for mechanisms of latency and for vaccine and antiviral drug development.

Transitional epithelial lesions of the ureter in renal transplant rejection.

The spectrum of ureteric lesions of human renal allografts, long attributed exclusively to postsurgical complications such as ischemia, has recently been shown to include the types of rejection seen in the kidney. Since the rejected ureter also exhibits transitional epithelial lesions that may impact on renal and ureteral function, we studied, by light, immunohistochemical, immunofluorescent, and electron microscopic techniques, ureters of 65 irreversibly rejected kidneys. Seven unused cadaver kidneys served as controls. Urothelial lesions, noticed in 57 of 65 ureters (88%), ranged from minimal basal vacuolization to complete sloughing with or without necrosis of the epithelial lining. Epithelial exfoliation was noticed in 31 cases (54.4%), and basal vacuolization, severe enough to produce cleavage of the epithelial junctions and thus create bullae, was noticed in 21 cases (36.8%). Immunofluorescent and immunoperoxidase stains, performed in 16 cases, were all positive for immunoglobulins but yielded varied results ranging from granular to linear staining, particularly in the region of the basal cells and the basement membrane. Electron microscopic findings confirmed the light microscopic alterations. By contrast, control ureters showed no lesions. Urothelial ureteric lesions might impede ureteral functions and result in obstruction or infection, thus compounding the consequences of renal allograft rejection. Moreover, elucidation of the pathophysiology of the process will advance the understanding of various cutaneous and transitional epithelial autoimmune conditions.