Biomedical subjects
Publications and source records attributed to A C Phillips.
Loss of the p53 tumor suppressor pathway contributes to the development of most human cancers. p53 is a nuclear protein that functions as a regulator of transcription. Significant advances have been made recently in our understanding of how p53 function is regulated and the mechanisms by which p53 mediates its effects.
Members of the E2F family of transcription factors play an important role in regulating the cell cycle, and their activity is often perturbed during the development of human malignancies. More recent work has shown that E2F-1 regulates apoptosis as well as proliferation, in part by stabilizing the p53 tumor suppressor, an important mediator of apoptosis. This has led to the suggestion that E2F-1 may function as a tumor surveillance mechanism, detecting aberrant proliferation and engaging apoptotic pathways to protect the organism from developing tumors.
Using competitive binding experiments, it was found that native type XI collagen binds heparin, heparan sulfate, and dermatan sulfate. However, interactions were not evident with hyaluronic acid, keratan sulfate, or chondroitin sulfate chains over the concentration range studied. Chondrocyte-matrix interactions were investigated using cell attachment to solid phase type XI collagen. Pretreatment of chondrocytes with either heparin or heparinase significantly reduced attachment to type XI collagen. Incubation of denatured and cyanogen bromide-cleaved type XI collagen with radiolabeled heparin identified sites of interaction on the alpha1(XI) and alpha2(XI) chains. NH(2)-terminal sequence data confirmed that the predominant heparin-binding peptide contained the sequence GKPGPRGQRGPTGPRGSRGAR from the alpha1(XI) chain. Using rotary shadowing electron microscopy of native type XI collagen molecules and heparin-bovine serum albumin conjugate, an additional binding site was identified at one end of the triple helical region of the collagen molecule. This coincides with consensus heparin binding motifs present at the amino-terminal ends of both the alpha1(XI) and the alpha2(XI) chains. The contribution of glycosaminoglycan-type XI collagen interactions to cartilage matrix stabilization is discussed.
The transcription factor E2F-1 induces both cell-cycle progression and, in certain settings, apoptosis. E2F-1 uses both p53-dependent and p53-independent pathways to kill cells. The p53-dependent pathway involves the induction by E2F-1 of the human tumour-suppressor protein p14ARF, which neutralizes HDM2 (human homologue of MDM2) and thereby stabilizes the p53 protein. Here we show that E2F-1 induces the transcription of the p53 homologue p73. Disruption of p73 function inhibited E2F-1-induced apoptosis in p53-defective tumour cells and in p53-/- mouse embryo fibroblasts. We conclude that activation of p73 provides a means for E2F-1 to induce death in the absence of p53.
The acetyltransferase p300 was first identified associated with the adenoviral transforming protein E1A, suggesting a potential role for p300 in the regulation of cell proliferation. Direct evidence demonstrating a role for p300 in human tumours was lacking until the recently publication by Gayther et al, which strongly supports a role for p300 as a tumour suppressor. The authors identify truncating mutations associated with the loss or mutation of the second allele in both tumour samples and cell lines, suggesting that loss of p300 may play a role in the development of a subset of human cancers.
The E2F family of transcription factors plays an essential role in promoting cell cycle progression, and one member of the family, E2F-1, is also capable of inducing apoptosis. We show here that E2F-1 can induce apoptosis by a death receptor-dependent mechanism, by downregulating TRAF2 protein levels and inhibiting activation of antiapoptotic signals including NF-kappa B. In this way, E2F-1 expression can lead to the sensitization of cells to apoptosis by a number of agents independently of p53. Deregulation of E2F-1 activity occurs in the majority of human tumors, and the ability of E2F-1 to inhibit antiapoptotic signaling may contribute to the enhanced sensitivity of transformed cells to chemotherapeutic agents.
p21Waf1/Cip1 is a major transcriptional target of p53 and has been shown to be one of the principal mediators of the p53 induced G1 cell cycle arrest. We show that in addition to the G1 block, p21Waf1/Cip1 can also contribute to a delay in G2 and expression of p21Waf1/Cip1 gives rise to cell cycle profiles essentially indistinguishable from those obtained following p53 expression. Arrest of cells in G2 likely reflects an inability to induce cyclin B1/cdc2 kinase activity in the presence of p21Waf1/Cip1, although the inefficient association of p21Waf1/Cip1 and cyclin B1 suggests that the mechanism of inhibition is indirect. Cells released from an S-phase block were not retarded in their ability to progress through S-phase by the presence of p21Waf1/Cip1, despite efficient inhibition of cyclin E, A and B1 dependent kinase activity, suggesting that p21Waf1/Cip1 is inefficient at inhibiting replicative DNA synthesis in vivo. Interestingly, significant numbers of cells released from the p21Waf1/Cip1 activated G2 block undergo endoreduplication, passing through another S-phase before undergoing mitosis. This supports a function of the mitotic kinases in both entry into mitosis, and also in preventing re-replication of DNA following S-phase and suggests a role for p21Waf1/Cip1 in coupling DNA synthesis and mitosis. Unlike p53, which induces apoptosis in these cells, extended expression of p21Waf1/Cip1 resulted in the expression of a senescent-like phenotype in these p53 null, pRB null tumor cells.
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OBJECTIVE: To evaluate the efficacy and tolerability of an oral preparation of iloprost, a prostacyclin analog, in patients with Raynaud's phenomenon (RP) secondary to systemic sclerosis (scleroderma). METHODS: A multicenter, randomized, parallel-group, placebo-controlled double-blind study was performed at university and community-based medical centers. Patients were randomly assigned to receive either 50 microg of iloprost orally twice daily or an identical gelatin-coated capsule containing placebo for 6 weeks. Outcome measures included average total daily duration of RP attacks, average number of RP attacks, and RP condition scored via a standardized daily diary. RESULTS: Three hundred eight patients with scleroderma (272 women, 36 men, mean age 49 years [range 18-80]) were enrolled. One hundred fifty seven were assigned to receive iloprost and 151 to receive placebo. One hundred forty-three patients in the iloprost group (91.1%) and 144 in the placebo group (95.4%) completed the 6-week treatment phase. Fifteen of these treated patients (8 iloprost, 7 placebo) failed to complete all of the followup visits. The mean reduction in the average duration of attacks from baseline to week 5-6 was 24.32 minutes in the iloprost group and 34.34 minutes in the placebo group (P = 0.569). Likewise, the mean reduction from baseline to week 5-6 in the daily frequency of attacks was 1.02 in the iloprost group and 0.83 in the placebo group (P = 0.459). The Raynaud's condition score, a patient-completed assessment of the severity of RP attacks, was reduced by 1.32 in the iloprost group and 1.00 in the placebo group (P = 0.323). The lack of significant difference between treatment groups did not change when a variety of factors, including use of other vasodilators, duration of disease, classification of scleroderma (limited versus diffuse), or number of baseline digital ulcers were taken into account. Premature withdrawal from the study due to adverse events occurred in 10 patients (6.4%) in the iloprost group and 3 (2.0%) in the placebo group (P = 0.058). CONCLUSION: Oral iloprost at a dosage of 50 microg twice daily is no better than placebo for management of RP secondary to scleroderma, either during 6 weeks of treatment or during 6 weeks of posttreatment followup.
The differentiation of articular from nonarticular complaints and inflammatory from noninflammatory disorders can usually be made by clinical evaluation. Laboratory and radiographic studies serve as adjuncts in the evaluation of musculoskeletal complaints; however, before ordering these studies, it is important to consider how the findings will affect diagnosis, prognosis, and treatment. An effective clinical evaluation is frequently sufficient to establish a differential diagnosis, focus further confirmatory testing, and consider appropriate treatment.
The family of E2F transcription factors have an essential role in mediating cell cycle progression, and recently, one of the E2F protein family, E2F-1, has been shown to participate in the induction of apoptosis. Cooperation between E2F and the p53 tumor suppressor protein in this apoptotic response had led to the suggestion that cell cycle progression induced by E2F-1 expression provides an apoptotic signal when placed in conflict with an arrest to cell cycle progression, such as provided by p53. We show here that although apoptosis is clearly enhanced by p53, E2F-1 can induce significant apoptosis in the absence of p53. Furthermore, this apoptotic function of E2F-1 is separable from the ability to accelerate entry into DNA synthesis. Analysis of E2F-1 mutants indicates that although DNA-binding is required, transcriptional transactivation is not necessary for the induction of apoptosis by E2F-1, suggesting that it may be mediated through alleviation of E2F-dependent transcriptional repression. These results indicate that E2F-1 can show independent cell cycle progression and apoptotic functions, consistent with its putative role as a tumor suppressor.
The E7 protein encoded by human papillomavirus type 16 shows transforming and immortalizing activities which are mediated, in part, through the interaction of the viral oncoprotein with the pRB protein family. This interaction is not solely responsible for E7 function, however, and other properties of E7, such as the interaction with basal transcription factors such as TBP, are likely to be of importance. We show here that three regions of the viral protein contribute to the interaction between E7 and TBP; the pRB-binding domain, the casein kinase II phosphorylation region and the C-terminal dimerization domain. Mutations within each region reduced the interaction of E7 with TBP in vitro, and simultaneous alterations within each of these regions completely abrogated binding. Unlike the pRB interaction, the association of E7 with TBP was enhanced following phosphorylation of E7 by casein kinase II, demonstrating a functional significance for phosphorylation of the viral protein.
Despite their propensity to cause toxicity, nonsteroidal anti-inflammatory drugs (NSAIDs) are routinely prescribed for older patients for painful musculoskeletal conditions, many of which are noninflammatory in nature. In some settings, simple analgesia with paracetamol (acetaminophen) or tramadol may be just as effective as NSAIDs. The benefits of therapy with NSAIDs must be weighed against their potential risks. With the anticipated growth of the elderly population, the economic implications of NSAID use in older patients are staggering. Estimates of the total cost of prescribing NSAIDs to the elderly must include the additional costs of gastroprotective agents, prophylaxis, laboratory monitoring, physician evaluations and interventions for adverse effects. Misoprostol may be cost effective as primary prophylaxis of NSAID-induced ulcer disease in some elderly patients. NSAIDs may reduce disability and improve quality of life, thereby offsetting their costs. To date, the direct, indirect and intangible cost of NSAIDs and cost offsets have not been systematically evaluated in the elderly. At this juncture, NSAIDs may be used judiciously in older patients, and misoprostol should be considered in high risk patients. Further studies to assess the economics of NSAIDs in the elderly population are warranted.
1. To determine whether dexfenfluramine is a substrate of cytochrome P450 2D6 (CYP2D6), its disposition has been studied in nine extensive (EM) and eight poor metabolizers (PM) of debrisoquine. 2. Following a 30 mg dose of dexfenfluramine hydrochloride, urine was collected in all subjects for 96 h post-dose and plasma samples were collected in 11 subjects (six EMs and five PMs). Dexfenfluramine and nordexfenfluramine were measured in urine by h.p.l.c. and in plasma by g.c. 3. Urinary recovery of dexfenfluramine was greater in PMs than EMs (4136 +/- 1509 micrograms vs 1986 +/- 792 micrograms; 95% CI of difference 926-3374; P < 0.05) whereas that of nordexfenfluramine was similar in both phenotypes (PM: 1753 +/- 411 micrograms vs 1626 +/- 444 micrograms). 4. Dexfenfluramine AUC was higher in PMs (677 +/- 348 micrograms l-1 h) than EMs 359 +/- 250 micrograms l-1 h). The apparent oral clearance of dexfenfluramine was greater in EMs than PMs (93.6 +/- 42.4 l h-1 vs 45.6 +/- 19.5 l h-1; 95% CI of difference 1.2-94.7; P < 0.05). The renal clearance was similar in both phenotypes (EMs: 5.88 +/- 2.83 l h-1; PMs 6.60 +/- 2.01 l h-1), indicating that the higher urinary recovery of dexfenfluramine in PMs reflects higher plasma concentrations, rather than phenotype differences in the renal handling, of dexfenfluramine. 5. The apparent nonrenal clearance of dexfenfluramine was substantially lower (P < 0.05; 95% CI of difference 3.0-94.1) in PMs (39.0 +/- 19.5 l h-1) than EMs (87.6 +/- 41.2 l h-1). 6. There was a significant inverse correlation (rs = 0.776 95% CI-0.31-0.94; n = 11; p = 0.005) between the debrisoquine metabolic ratio and the apparent nonrenal clearance of dexfenfluramine. 7. PMs had a higher incidence of adverse effects (nausea and vomiting) than EMs. 8. In conclusion, the metabolism of dexfenfluramine is impaired in PMs. Thus CYP2D6, the isoenzyme deficient in poor metabolizers of debrisoquine, must catalyse at least one pathway of dexfenfluramine biotransformation.
A method to determine the concentration of dexfenfluramine and its active metabolite nordexfenfluramine in human urine from healthy volunteers is described utilising a high-performance liquid chromatographic procedure with liquid-liquid extraction and ultraviolet detection. Analytes are measured after extraction of alkalinised urine with diethyl ether and subsequent back extraction with 0.5 M H2SO4 and with chromatography performed on a reversed-phase C18 column, using a mobile phase of acetonitrile-50 mM K2HPO4 (25:75, v/v) (flow-rate 1.3 ml/min) and ultraviolet detection at 210 nm. The sensitivity of the technique (10 ng/ml) is appropriate to measure both parent drug and metabolite in urine in humans for up to 5 days after a single 30-mg dose. The method is selective, reproducible (within- and between-day coefficient of variation ranged from 4.2 to 15%) and accurate (bias less than 8%) and thus suitable for dexfenfluramine pharmacokinetic investigations.
A biotin-labelled DNA probe was used to detect the presence of bacteria carrying plasmid DNA and to study the effect of ciprofloxacin and nalidixic acid on plasmid carriage. The colour intensity generated in dot-blot hybridization tests was measured with a computer-controlled image analysis system and was shown to be related to the number of plasmid copies present. Ciprofloxacin, at about 0.5 x MIC, reduced the plasmid copy number in a bacterial population, while nalidixic acid, used at a similar ratio to MIC, had a much smaller effect. The method could potentially be used to study the effect of antibiotics on plasmid replication and curing in any bacterial system for which a suitable DNA probe is available.
The effects of thymosin and lipopolysaccharide (LPS) on cyclic AMP levels in lymphocytes were evaluated using three independent assays which included adenine prelabeling, protein kinase binding, and radioimmunoassay. All three assays proved to be both sensitive and accurate in assessing relative changes in lymphocytes after incubation in vitro with various agents. The assays confirmed that basal and stimulated levels of cyclic AMP depended on the origin of the lymphocyte population. Each of the three techniques demonstrated that pyrogen-free bovine thymosin fraction 5 did not elevate thymocyte cAMP levels. In contrast, it was found that lipopolysaccharide (lps) significantly elevated cAMP levels in both spleen and thymus lymphocytes. These studies indicate that assays for measuring the activity of thymic extracts in which the intracellular levels of cyclic nucleotides are a criterion for activity are only valid if the preparations are not contaminated with endotoxins.