No abstract

Conditional expression of wild-type (wt) p53 protein in a glioblastoma tumor cell line has been shown to be growth inhibitory. We have now more precisely localized the position in the cell cycle where growth arrest occurs. We show that growth arrest occurs prior to or near the rici on point in late G1 phase of the cell cycle. The effect of wt p53 protein on the expression of four iediate-earl genes (c-FOS, c-JUN, JUN-B, and c-MYC), one delayed-early gene (ornithine decarboxylase), and two late-G1/S-phase genes (B-MYB and DNA polymerase a) was also examined. Of this subset of growth response gene, only the expression ofB-MYB and DNA polymerase a was slgicantly repressed. The possibility that decreased expression of B-MYB may be an important component of growth arrest mediated by wt p53 protein is diss. Cell cycle controls that regulate the orderly flow of cells in and out of G1 phase are the main determinant of the rate of postembryonic cell proliferation (8, 9). Two major control points have been defined in animal cells. One control point appears to operate in early G1 phase and allows cells to exit the cell cycle and enter a nonproliferative state of arrest termed Go (8,9). The second control point, the restriction point (R-point) occurs in late G1 phase (8, 10). Time-course experiments indicate that protein synthesis is required throughout early G1 phase for cells to pass the R-point and become committed to enter S phase and initiate DNA synthesis. The R-point is thus defined as the time after which inhibition of protein synthesis fails to inhibit entry into S phase (8, 10).We have shown that induction ofwt p53 protein expression in a human glioblastoma tumor cell line can inhibit cell cycle progression (5). In this model, growth inhibition was associated with a significant decrease in the steady-state mRNA levels of the replication-dependent histone H3 gene (5), which is known to be coordinately regulated with the onset of DNA replication (11), and the replication-independent proliferating cell nuclear antigen (PCNA) gene (12). On the contrary, the steady-state mRNA levels for the genes encoding endogenous p53, (32-microglobulin, (-actin, and thymidine kinase were not found to be significantly affected (5,12).In the present communication, we have more precisely localized the position in the cell cycle where growth suppression occurs, and we have examined the effect of induction of wt-p53 protein on the expression of seven additional growth response genes. Evidence is presented that growth suppression mediated by wt p53 protein occurs prior to or near the R-point in late G1 phase ofthe cell cycle and that only a subset of late-G1/S-phase growth response genes are affected. MATERIALS AND METHODSCell Lines and Culture Conditions. The parental human glioblastoma cell line T98G and the sublines GM47.23 and Mdel 4A were cultured in Earle's minimal essential medium containing 10% fetal calf serum (GIBCO) at 370C as described (5, 12). The Mdel 4A subline was derived by transfection with the pMdel plasmid (4) ...

The p53 gene is a frequent target of mutation in a wide variety of human cancers. Previously, it was reported that conditional expression ofwild-type p53 protein in a cell line (GM47.23) derived from a human glioblastoma multiform tumor had a negative effect on cell proliferation. We have now investigated the effect that induction of wild-type p53 protein in this cell line has on the expression of the proliferating-cell nuclear antigen gene. The proliferating-cell nuclear antigen gene encodes a nuclear protein that is an auxiliary factor of DNA polymerase 8 and part of the DNA replication machinery of the cell. We show that inhibition ofcell cycle progression into S-phase after induction ofwild-type p53 protein is accompanied by selective down-regulation of proliferating-cell nuclear antigen mRNA and protein expression. (14). Thus these studies support the hypothesis that the inhibitory effect of wt-p53 on oncogene-mediated transformation may be directly linked to its growth-suppressing activity and that this function can be inactivated by mutation.Direct experimental evidence for a growth-suppressing function of wt-p53 was provided by the demonstration that conditional expression of wt-p53 protein in a cell line derived from a human glioblastoma multiform tumor blocks cell cycle progression (15). In this system, inhibition of cell cycle progression was associated with a marked decrease in histone H3 mRNA expression, an S-phase marker gene whose regulation is tightly linked to DNA replication. This finding suggested that the wt-p53 protein induced in these cells may specifically effect a function (or functions) required for progression from G1 into S phase. To gain further insight into the possible mechanism(s) of growth suppression induced by wt-p53, we studied the effect of wt-p53 protein on the expression of the gene encoding proliferating-cell nuclear antigen (PCNA Plasmids and Probe Preparation. The probes used for hybridization were from plasmids digested with the appropriate restriction enzyme and followed by gel purification. They include a human p53 cDNA Xba I fragment of p53H (21), a /3-actin BamHI fragment of pHFf3A-1 (22), a human histone H3 EcoRI fragment of pFO422 (23), a human PCNA cDNA fragment of pPCNA-G3, a human thymidine kinase (TK) cDNA fragment ofpTK11 (kind gifts ofRenato Baserga, Abbreviations: wt, wild type; PCNA, proliferating-cell nuclear antigen; Dex, dexamethasone; TK, thymidine kinase. tTo whom reprint requests should be addressed at: 3400 North Broad Street, Philadelphia, PA 19140. 1958 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Overexpression of wild-type p53 protein has been shown to induce arrest in the G, stage of the cell cycle and to transactivate expression of the gene that encodes the 21-kDa Wafl/Cipl protein, a potent inhibitor of cyclin-dependent kinase activity. p53-dependent G, arrest is accompanied by decreased expression of the B-myb gene, a relative of the c-myb cellular oncogene. In this study we show that B-myb expression is required for cells to progress from G, into S phase and that high levels of ectopic B-myb expression uncoupled from cell cycle regulation rescues cells from p53-induced G, arest even in the presence of Wafl/Cipl transactivation and inhibition of cyclin E/Cdk2 kinas activity. Cotransfection experiments with p53 expression plaids and expression plasmids encoding in-frame deletion mutations in B-myb coding sequences indicate that the DNA-binding domain of the B-Myb protein is required for this activity. These results provide evidence of a bypass of p53-induced Wafl/Cipl-mediated cell cycle regulatory pathways by a member of the myb oncogene family.

No abstract

One hundred fifty-one patients with primary biliary cirrhosis (PBC) grouped into four strata based on entry serum bilirubin (<2 mg/dL vs. 2 mg/dL or greater) and liver histology (stages I, 1 1 vs. stages 111, IV-Ludwig criteria) were randomized within each stratum to ursodiol or placebo given in a single dose of 10 to 12 mgkg at bedtime for 2 years. Placebo-(n = 74) and ursodioltreated (n = 77) patients were well matched at baseline for demographic and prognostic factors. Ursodiol induced major improvements in biochemical tests of the liver in strata 1 and 2 (entry bilirubin <2), but had less effect on laboratory tests in patients with entry serum bilirubin of 2 2 (strata 3 and 4). Histology was favorably affected by ursodiol in patients in strata 1 and 2 but not in strata 3 and 4. Ursodiol enrichment in fasting bile obtained at the conclusion of the trial was approximately 40% and comparable in all strata. Thus, differences in ursodiol enrichment of the bile acid pool do not explain better responses of laboratory tests and histology found in patients with less advanced PBC. Patients treated with ursodiol tended to develop a treatment failure less frequently than those who received placebo, particularly in strata 1 and 2 (ursodiol42%, placebo WO, P = .078). Development of severe symptoms (fatigue/pru-Abbreviations: PBC, primary biliary cirrhosis; M-W, Mann-Whitney. From the 'University of Texas Southwestern Medical Center a t Dallas, Dallas, Tx; '

No abstract

In the present study, we investigated the effects of the Sonic hedgehog (Shh) protein on porcine oocyte maturation and early embryo development. Immunohistochemistry showed activation of Shh signalling in cumulus-oocyte complexes (COCs), as reflected by Patched (Ptc), Smoothened (Smo) and Gli1 expression in oocytes, cumulus cells and granulosa cells, particularly those of small follicles (<2 mm in diameter). Western blot analysis showed Smo expression in COCs and in denuded oocytes derived from small and medium (3-7 mm)-sized follicles. Small follicles contained the highest concentration of Shh in follicular fluid compared with medium-sized and large (>7 mm in diameter) follicles. Supplementation with Shh (0.5 or 1 microg mL(-1)) enhanced oocyte maturation compared with the control group (92.4% and 90.4% v. 81.9%, respectively; P < 0.05). This effect was reversed by the simultaneous addition of cyclopamine (1-2 microm), an Shh inhibitor. Similar to intact COCs, denuded COCs showed enhanced maturation following Shh supplementation. Furthermore, cyclin B1 content, extracellular signal-regulated kinase 1/2 phosphorylation, intracellular calcium release, blastocyst rate and total cell numbers were greater (P < 0.05) in oocytes matured in the presence of 0.5 and 1 microg mL(-1) Shh compared with control oocytes. The findings of the present study provide the first evidence that the Shh signalling pathway is active, or at least partially activated, in the porcine ovary and is likely to promote oocyte cytoplasmic and nuclear maturation, as well as subsequent in vitro development, although the underlying mechanisms remain to be elucidated.