Qi X. Yuan

Pathogenesis of mallory body formation: Studies using the drug-primed mouse model

Abstract Mallory bodies (MB) are formed in preneoplastic foci in the drug primed mouse model. These foci expand when the drug is refed. To better understand this phenotypic change in hepatocytes, the livers response to refeeding the drug was studied for transcriptional and post-translational changes. The post-translational change, hyperphosphorylation of MBs, was associated with changes in gene expression and activation of transcription regulation factors. Hyperphosphorylation of MBs appeared on the second day of refeeding the drug, the same time interval when MB formation began. Prior studies using this model showed an increase in the mitotic index and an increase in cytokeratin proteins at this time interval. In the present study, cytokeratin mRNA increased significantly, compared to the 0 day base line. Likewise, c-jun mRNA increased significantly at the same time interval. Previous studies on mice fed the drug for 5 months showed a significant increase in c-fos mRNA. The binding activity of AP-1 consensus oligonucleotide was significantly increased on the 5th day of refeeding. Taken together, the data support the conclusion that the MB phenotype change expands as the result of a response to stimulation of liver cell proliferation.

Mouse model of hepatocellular hyperplastic nodule formation characterization of mRNA expression

Abstract In previous studies a mouse model of hepatocellular tumorigenesis was developed. In this model of chronic griseofulvin feeding, preneoplastic foci developed over 5 months and numerous hyperplastic nodules with a few hepatocellular carcinomas developed after 10 months. In a subsequent study where 5- and 16-month livers were tested, the immediate early gene c-fos mRNA and transcription factor AP-1 were activated as well as was NFκB at both time intervals. However, the PPARα and RXRα genes were down-regulated. The evidence indicated that immediate early genes were involved in the promotion of tumor formation and that the direct hyperplasia pathway of regeneration was suppressed. To further characterize the involvement of the immediate early gene expression as well as other genes involved in the preneoplastic process we measured mRNA for c-jun, c-myc, hepatocyte growth factor activator (HGF-A), TGFβRII, γ-glutamyl transpeptidase (GGT), cytokeratin (CK8), ubiquitin (UB) and cellular transglutaminase (TG). The data indicated that c-jun an immediate early gene and c-myc, a delayed early gene were up-regulated at 5 and 16 months of feeding, both when preneoplastic foci appeared and when hyperplastic nodules developed. However, HGF-A was down-regulated at both time intervals. TGFβRII was up-regulated, as was GGT, CK8, TG and UB. GGT up-regulation was the only progression seen in gene expression at 16 months. It is concluded that a complex of cell proliferation and cell maintenance genes are involved in tumorigenesis in the mouse model of tumor promotion.

Newborn rabbit gastric smooth muscle cell culture : EGF and TGF-α are potent mitogens

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are two in a family of growth-promoting peptides for many gastrointestinal epithelia. This study was designed to assess their mitogenic effect on cultured gastric myocytes and to characterize specific EGF receptors on these cells. Single myocytes were isolated from newborn rabbit gastric fundus and placed into tissue culture. The composition of the culture at confluence as assessed by immunostaining with smooth muscle actin-specific monoclonal antibody (CGA7) was > 95% myocytes. To assess the effect of putative growth factors, freshly isolated myocytes were incubated in Dulbeccos modified Eagles (DME) medium containing 1% fetal bovine serum in the presence or absence of growth factors. After 6 days, cells were incubated in serum-free medium with [3H]thymidine (1 microCi/ml) in the continued presence or absence of growth factors. After 24 h, EGF and TGF-alpha but not insulin-like growth factor I (IGF-I) induced a dose-dependent increase in [3H]thymidine incorporation. Ten nanomolar EGF or TGF-alpha increased [3H]thymidine incorporation more than sixfold over control. EGF was more potent than was TGF-alpha, with apparent median effective dose (ED50) values of 64 +/- 14 pM and 166 +/- 62 pM (p < 0.05), respectively. EGF bound to cultured myocytes with Kd = 7.6 +/- 1.8 nM and Bmax = 27 +/- 11 pmol/mg DNA or 440,000 receptors/cell. TGF-alpha competed for binding at these receptors. Although IGF-I did not stimulate thymidine incorporation, specific high-affinity receptors for IGF-I were detected on gastric myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cell culture on rabbit colonic smooth muscle bradykinin receptors

The aim of this study was to assess the effect of cell culture on the bradykinin receptor of rabbit colon myocytes. In longitudinal muscle strips prepared from distal colon, bradykinin stimulated dose-dependent contraction that was 62% of the maximal response to bethanecol. At 4 degrees C, [3H]bradykinin binding to fresh muscle homogenates from the distal colon was time dependent, saturable, and linearly related to tissue concentration. Specific binding of 0.6 nmol/L [3H]bradykinin was 80% +/- 2% of total binding. In competitive binding studies, Hill coefficients approached unity, suggesting the presence of a single class of receptors. The order of potency was bradykinin greater than [D-Phe7]bradykinin much greater than des-Arg9, [Leu8]bradykinin, which is consistent with results of a B2 receptor subclass. Colon myocytes from the longitudinal muscle layer achieved confluence and were harvested for studies after 12-14 days in culture. Bradykinin receptors were of high affinity [disassociation constant (Kd) = 672 pmol/L] and numbered 10,217 +/- 2567/cell. To show that the receptors on cultured myocytes were functional, the effect of bradykinin was measured (a) on intracellular calcium concentration using Fura 2 and (b) on prostaglandin E2 concentration in the culture media using radioimmunoassay. In cells grown to confluence on cover slips and preloaded with Fura 2, bradykinin stimulated the threshold response at 1 nmol/L and maximal response (increased intracellular calcium concentration from 229 to 633 nmol/L) at 1 mumol/L. Bradykinin, 100 nmol/L, increased Prostaglandin E2 in the culture media threefold. In summary, colon myocytes express functioning bradykinin receptors, which, unlike muscarinic receptors, persist in culture. Bradykinin appears to be a suitable agonist for studies of receptor-mediated intracellular events in cultured colon myocytes.