RIP1 links inflammatory and growth factor signaling pathways by regulating expression of the EGFR.

There is considerable interest in understanding how inflammatory responses influence cell proliferation and cancer. In this study, we show that the receptor-interacting protein (RIP1), a critical mediator of inflammation and stress-induced NF-kappaB activation, regulates the expression of the epidermal growth factor receptor (EGFR). Mouse embryo fibroblasts (MEFs) derived from RIP1 knockout mice express very high levels of the EGFR. Reconstitution of RIP1(-/-) MEFs with RIP1 results in a lowering of EGFR levels. RIP1 influences EGFR at the mRNA level by regulating the EGFR promoter. Expression of RIP1 inhibits the EGFR promoter. RIP1 downregulates EGFR expression by interfering with the function of Sp1, which is a key activator of EGFR transcription. RIP1 suppresses Sp1 activity and overexpression of Sp1 reverses RIP1-mediated repression of the EGFR promoter. RIP1 is present both in the cytoplasm and in the nucleus. RIP1 coimmunoprecipitates with Sp1 in vivo and binds directly to Sp1 in vitro. A RIP1 mutant lacking the death domain fails to suppress Sp1 activity and the EGFR promoter, suggesting a critical role for the RIP1 death domain in EGFR regulation. Thus, our study identifies a new link between inflammatory and growth factor signaling pathways mediated by RIP1 and provides insight into the mechanism used by RIP1 to regulate EGFR levels.

Two implants for all edentulous mandibles.

Complete dentures have always been a poor substitute for natural teeth. Mandibular complete dentures frequently cause pain and discomfort, accelerated residual bone resorption, while failing to restore effective chewing. The provision of two implants to stabilise the mandibular complete denture can result in significant improvements.

Regression of human breast tumor xenografts in response to (E)-2'-deoxy-2'-(fluoromethylene)cytidine, an inhibitor of ribonucleoside diphosphate reductase.

(E)-2'-Deoxy-2'-(fluoromethylene)cytidine (MDL 101,731) is a mechanism-based inhibitor of ribonucleoside diphosphate reductase (J. Stubbe, personal communication), an enzyme involved in DNA synthesis and therefore a potential target for cancer chemotherapy. In the present report, we show that MDL 101,731 inhibits the proliferation of several human breast cancer cell lines, including the estrogen-dependent cell line, MCF-7, and the estrogen-independent cell lines MDA-MB-231, MDA-MB-468, and MDA-MB-435 in vitro at nanomolar concentrations (50% inhibitory concentration, 15-26 nM). Administration of MDL 101,731 caused marked regression of tumors which formed after s.c. inoculation of all four of the cell lines in athymic (nude) mice. MDA-MB-231 tumors were found to be most sensitive to MDL 101,731 with a 90-100% cure rate at doses of MDL 101,731 between 2 and 20 mg/kg, given as once daily i.p. injections, 5 days/week for as little as 3 weeks. Almost complete cessation of MDA-MB-231 tumor growth was obtained with a dose of 0.5 mg/kg MDL 101,731 following the same dosing regimen. MDA-MB-468, MDA-MB-435, and MCF-7 tumors were not as sensitive as MDA-MB-231, but tumor regression of 50, 65, and 80%, respectively, was obtained after 5-6 weeks of treatment. The effects of MDL 101,731 on spontaneous metastasis of MDA-MB-435 cells from the mammary fat pad to the lung was also examined, and it was found that the number of lung metastases was significantly decreased if mice received MDL 101,731 while the primary tumors were growing and after primary tumors were surgically excised. Additionally, preliminary evidence raises the possibility that MDL 101,731 may induce apoptosis in MDA-MB-231 tumors. Our data suggest that the use of MDL 101,731 for the treatment of breast cancer and possibly other solid tumors should be pursued.

Regulation of proline-rich protein gene expression by cyclic AMP in primary cultures of hamster parotid glands.

Proline-rich proteins (PRPs) constitute a group of unusual salivary proteins encoded by tissue-specific multigene families which can be dramatically induced (20- to 70-fold) in vivo in rats, mice and hamsters by treatment with the beta-agonist isoproterenol. Addition of dibutyryl cyclic AMP (dbcAMP) or forskolin to hamster parotid gland primary cultures resulted in a large increase (15- to 30-fold) in PRP mRNA levels. The same time-course and levels of induction of PRP mRNA by dbcAMP and isoproterenol were found in primary cultures, indicating that both effectors act through the same mechanism. Induction by isoproterenol, but not by dbcAMP or forskolin, was blocked by the beta-antagonist propranolol. Incorporation of [3H]proline into PRPs was stimulated in primary cultures by all three effectors. The greatest increase in proline incorporation was in the [3H]PRPs recovered in the culture medium of induced cells. These studies demonstrate that cAMP or agents which increase intracellular cAMP levels increase PRP gene expression in primary cultures of parotid glands. Pretreatment of the cells with cycloheximide blocked the induction of PRP mRNAs which indicates that the synthesis of a trans-acting factor may be necessary for transcriptional activation of the PRP genes. alpha-Amylase mRNA, another tissue-specific gene product, was not significantly affected by cycloheximide treatment.

Crystal structure of human cyclin-dependent kinase 2 in complex with the adenine-derived inhibitor H717.

Cyclin-dependent kinases (CDKs) are regulatory proteins of the eukaryotic cell cycle. They act after association with different cyclins, the concentrations of which vary throughout the progression of the cell cycle. As central mediators of cell growth, CDKs are potential targets for inhibitory molecules that would allow disruption of the cell cycle in order to evoke an antiproliferative effect and may therefore be useful as cancer therapeutics. We synthesized several inhibitory 2,6,9-trisubstituted purine derivatives and solved the crystal structure of one of these compounds, H717, in complex with human CDK2 at 2.6 A resolution. The orientation of the C2-p-diaminocyclohexyl portion of the inhibitor is strikingly different from those of similar moieties in other related inhibitor complexes. The N9-cyclopentyl ring fully occupies a space in the enzyme which is otherwise empty, while the C6-N-aminobenzyl substituent points out of the ATP-binding site. The structure provides a basis for the further development of more potent inhibitory drugs.

Irreversible inhibition of S-adenosylmethionine decarboxylase in Plasmodium falciparum-infected erythrocytes: growth inhibition in vitro.

Blocking spermidine and spermine synthesis in Plasmodium falciparum-infected erythrocytes with irreversible inhibitors of S-adenosylmethionine decarboxylase (AdoMet DC; EC 4.1.1.50), prevented the growth of the parasite in vitro. The most potent of these compounds, MDL 73811, inhibited growth of chloroquine-sensitive and -resistant strains of P. falciparum equally, with an IC50 of 2-3 microM. Other structurally related compounds also inhibited parasite proliferation, but to a lesser degree, determined apparently by their potency for inhibition of AdoMet DC. The growth inhibition by MDL 73811 could be alleviated by incubating infected erythrocytes with spermidine and spermine, but not putrescine. Parasites treated with the drug were arrested at the trophozoite stage of the erythrocytic cycle and had putrescine levels which were elevated by about 3- to 4-fold. Treatment of crude extracts of purified parasites with 1 microM MDL 73811 inhibited AdoMet DC activity by greater than 90%. These biochemical changes in P. falciparum-infected cells were consistent with AdoMet DC inhibition being the primary effect of MDL 73811 treatment.

Disruption of Plasmodium falciparum-infected erythrocyte cytoadherence to human melanoma cells with inhibitors of glycoprotein processing.

Adherence of Plasmodium falciparum-infected erythrocytes (IE) to the venular endothelium in brain and other organs is characteristic of cerebral malaria, an often fatal complication in infected individuals. It has been shown that cytoadherence may be mediated through interaction of IE with glycoproteins on host target cell surfaces, including CD36 (GPIV), intercellular adhesion molecule-1 (ICAM-1), and thrombospondin. Inhibitors of glycoprotein synthesis and processing were tested for their abilities to decrease IE adherence to C32 human melanoma cells. The alpha-glucosidase inhibitor, castanospermine, was effective in disrupting cytoadherence in vitro when incubated with C32 cells (IC50 = 600-700 microM). Castanospermine-6-butyrate was even more effective than the parent compound (IC50 = 9 microM) in disrupting cytoadherence. The mannosidase inhibitors, swainsonine and deoxymannojirimycin, had no effect on cytoadherence at concentrations up to 2 mM. No effect on cytoadherence was observed when the glucosidase and mannosidase inhibitors were incubated with IE rather than the C32 cell cultures. The level of CD36 on the C32 cell surface was decreased as measured by fluorescence-activated cell sorting (FACS) analysis with the same inhibitors which inhibited cytoadherence. Cells labeled with fluorescein isothiocyanate (FITC) OKM5 monoclonal antibody, which recognizes CD36 and disrupts cytoadherence, showed decreased fluorescence when treated with tunicamycin and castanospermine-6-butyrate but not when treated with swainsonine or deoxymannojirimycin. ICAM-1 levels, as measured by surface labeling of C32 cells with FITC CD54 monoclonal antibody, were decreased in cells treated with tunicamycin. However, incubation of cells with castanospermine-6-butyrate or deoxymannojirimycin decreased cell surface ICAM-1 levels only slightly. These findings suggest that (1) in C32 cells, levels of cell surface CD36, and not ICAM-1, change proportionally to the level of cytoadherence; (2) drugs which can affect the carbohydrate moiety of cellular glycoproteins decrease cytoadherence of IE to C32 cells; and (3) protection against the development of cerebral malaria may be possible with inhibitors of glycoprotein biosynthesis.

Clomiphene analogs with activity in vitro and in vivo against human breast cancer cells.

Six hundred triphenylethylenes were assayed for antiproliferative activity against MCF-7, LY2, and MDA-MB-231 breast cancer cells using sulforhodamine B dye to measure proliferation. Here we report on just 63 of the compounds, mostly clomiphene analogs, with substitutions on the alpha' or beta ring, at the vinyl position or in the side chain, of which 23 were active, as defined by antiproliferation IC50 values < or =1 microM. Activity profiles showed that 23 and 11 analogs were active toward MCF-7 and LY2, respectively, but none were active against MDA-MB-231. The IC50 values of tamoxifen were 2.0 microM against MCF-7 and 7.5 microM against LY2 and MDA-MB-231. Estradiol reversed antiproliferative activities of several E isomers but not their Z isomer counterparts. Clomiphene side chain analogs 46 [(E)-1-butanamine, 4-[4-(2-chloro-1,2-diphenylethenyl) phenoxy]-N,N-diethyl-dihydrogen citrate (MDL 103,323)] and 57 [(E)-N-[p-(2-chloro-1,2-diphenylvinyl) phenyl]-N,N-diethylethylenediamine dihydrogen citrate (MDL 101,986)] were 4- to 5-fold more effective than tamoxifen. Methylene additions up to (-CH2-)12 in the clomiphene side chain showed that analog 46 [(-CH2-)4 side chain] had maximal antiproliferative activity, binding affinity, and inhibition of transcription of an estrogen response element luciferase construct in transfected MCF-7 cells. Intraperitoneal administration of 46 or 57 inhibited progression of MCF-7 breast tumor xenografts in nude mice with ED50 values of <0.02 mg/mouse/day. Both analogs may hold promise for treating ER positive breast cancer and are of interest for further development.

Depletion of estrogen receptor in human breast tumor cells by a novel substituted indole that does not bind to the hormone binding domain.

Steroidal antiestrogens appear to have at least two major modes of action in breast cancer cells, direct antagonism of estrogen binding to its receptor and depletion of estrogen receptors (ER) due to inhibition of dimerization of the receptor and a resultant destabilization of the receptor protein. In a search for other classes of compounds which would act as dimerization inhibitors, a novel substituted indole (8-{2-[1-(4-chlorobenzoyl)-5-hydroxy-2-methyl-1H-indol-3-yl]-acetylamino} octanoic acid butyl-methyl amide, MDL 101,906) was synthesized. Binding of the ER to its consensus response element (ERE) was apparently decreased in nuclear extracts from MCF-7 human breast cancer cell treated with MDL 101,906. This decreased binding was found to be due to depletion of ER based on direct measurement of ER using an enzyme-linked immunoassay. Other transcription factors were apparently unaffected by MDL 101,906 treatment. Whereas depletion of ER with a steroidal antiestrogen was almost complete after 3 h of treatment of MCF-7 cells, the effect of MDL 101,906 took significantly longer to occur, suggesting a fundamental difference in the mechanisms of action of the two drugs. This was also evident in the lack of binding of MDL 101,906 to the hormone binding domain of ER. MDL 101,906 treatment also caused depletion of ER mRNA in MCF-7 cells. Depletion of ER mRNA was noted by 3 h of drug treatment and was apparently almost complete after 24 h of treatment. Depletion of ER from MCF-7 cells led to a dose-dependent decrease in the expression of luciferase by an ERE-driven luciferase reporter gene assay system. The mechanism of MDL 101,906 appears to be unique and additional studies with this chemical class seem to be warranted to assess the potential for therapeutic utility.

Characterization of the adhesion of soft lining materials to poly (methyl methacrylate).

The adhesion of nine soft lining materials to poly (methyl methacrylate) has been characterized in the laboratory using peeling tests. Applying the energy theory of fracture to an elastic film peeling from a rigid substrate, this method gives a direct measure of the work of detachment.

Characterization of the rupture properties of denture soft lining materials.

Rupture properties of soft lining materials are characterized by their resistance to tearing. The energy necessary for their rupture depends upon rate of deformation, temperature, and conditions of storage or use. After storage in water for six months, some silicone rubber materials are no stronger than irreversible hydrocolloid materials.

Water absorption and water solubility of soft lining materials for acrylic dentures.

Soft lining materials undergo two processes when immersed in water. Plasticizers and other soluble materials are leached into the water, and water is absorbed by the polymer. The balance between these two processes affects both the compliance and dimensional stability of the materials.

The prevalence and significance of yeasts in persons wearing complete dentures with soft-lining materials.

Fifty-three persons wearing soft-lined mandibular dentures and heat-cured acrylic-resin maxillary dentures were studied, using imprint cultures, to determine the isolation frequency and density of colonization of denture and mucosal surfaces by yeasts. Yeasts were isolated from 35 (66%) of the persons studied. Nine species of Candida and one each of Trichosporon and Saccharomyces were identified. Candida albicans, occurring either alone or together with another strain, was identified in 66% of the isolates and was associated with a higher mean density/cm2 than that of other strains. An association between the method of denture cleaning, denture hygiene, and smoking habits and the isolation of yeasts was demonstrated, but a similar association could not be demonstrated with the sex of the person, denture-wearing habits, type and condition of the soft lining, or the clinical appearance of the mandibular denture-bearing mucosa. Although yeasts are more likely to colonize soft-lining materials than the fitting surface of conventional lower dentures, their presence did not significantly affect the soft-lining material. Further, the increased isolation of yeasts on the fitting surface of the soft-lined mandibular denture was not associated with an increased incidence of inflammatory changes in the mandibular denture-bearing mucosa.

A functional membrane repair system in Duchenne muscular dystrophy fibroblasts.

Experiments have been performed to determine if fibroblasts from patients with Duchenne muscular dystrophy (DMD) are defective in a process of membrane repair. Normal and DMD fibroblasts were treated with phospholipase C from Clostridium perfringens to degrade plasma membrane phosphatidylcholine, and then phosphatidylcholine synthesis was measured as the incorporation of [3H] choline into lipid. Phosphatidylcholine synthesis was stimulated by phospholipase C treatment to a similar extent in normal and DMD fibroblasts. The activity of CTP: phosphocholine cytidylyltransferase, the enzyme regulating phosphatidylcholine synthesis in phospholipase C-treated mammalian cells, was also stimulated to the same extent in both cell types. The subcellular location of the cytidylyltransferase was changed by phospholipase C treatment from mostly cytosolic to mostly particulate in both normal and DMD fibroblasts. It appears, therefore, that at least one type of membrane repair system functions normally in DMD fibroblasts.

Decreased vascular endothelial growth factor expression associated with tumor regression induced by (E)-2'-deoxy-2'-(fluoromethylene)cytidine (MDL 101,731).

The effect of the antitumor drug MDL 101,731 [(E)-2'-deoxy-2'-(fluoromethylene)cytidine] on tumor growth and on steady-state vascular endothelial growth factor (VEGF) mRNA levels in MDA-MB-231, PC-3, MCF-7, and HT-29 human tumor xenografts grown in nude mice was examined, using quantitative in situ hybridization. MDL 101,731 caused regression of MDA-MB-231 and PC-3 tumor xenografts, but only inhibition of growth (without regression) of MCF-7 xenografts. The drug caused inhibition of growth of HT-29 xenografts at low doses, and regression at high doses. When treatment with MDL 101,731 led to tumor regression, VEGF mRNA levels were decreased. When treatment led only to inhibition of growth, there was no significant change in VEGF mRNA. Further examination of the tumor xenografts revealed that elevated VEGF mRNA was associated with hypoxic zones surrounding areas of necrosis in the tumors, and that the drop in VEGF mRNA observed in tumors from mice treated with MDL 101,731 correlated with a loss of zones of necrosis. In contrast, treatment with cisplatin led to either an increase (PC-3) or no change (MDA-MB-231) in VEGF mRNA levels, and no loss of necrotic zones. Quantitative analysis of changes in VEGF mRNA levels was supported by immunohistochemical analysis of VEGF protein in the same tumor specimens. In vitro, MDL 101,731 was a potent inhibitor of VEGF secretion in cells exposed to hypoxia, whereas there was no effect of cisplatin on VEGF secretion by three of the four cell lines tested. These findings suggest that inhibition of VEGF expression by MDL 101,731 may distinguish this compound from other classes of cytotoxic agents, such as cisplatin.

Effect of prefabricated bar design with implant-stabilized prostheses on ridge resorption: a clinical report.

This investigation was concerned with the resorption of the posterior mandibular residual ridge in patients wearing mandibular overdentures supported by either parallel-sided bars (rigid joint) or oval straight bars (resilient joint) on two activated implants. Rotational tomographs were taken shortly after implant placement and up to 8 years later. Using proportional measurement, the area of residual ridge was measured in bilateral posterior areas. Patients rehabilitated with implant-stabilized mandibular overdentures demonstrated posterior mandibular residual ridge resorption at low rates, which were not significantly influenced by the design of the prefabricated bar.

Oral health impact on daily performance in patients with implant-stabilized overdentures and patients with conventional complete dentures.

This cohort study (n = 83) investigated whether patients with implant-stabilized overdentures would demonstrate less impact on daily life, would have less difficulty in the mastication of different types of food, and would generally be more satisfied than patients with conventional complete dentures. The groups were comparable for gender, age of dentures, and duration of edentulism. The patients were interviewed using a questionnaire, which included the Oral Impacts on Daily Performances (OIDP) sociodental indicator. Patients with implant-stabilized overdentures were more satisfied with the comfort of their dentures, could eat a wide range of food items with less difficulty, and experienced less impact on daily life than patients with conventional complete dentures. The findings of this study support the need to consider implant-stabilized overdentures in the treatment of edentulous patients.

The effects of prefabricated bar design on the success of overdentures stabilized by implants.

Two groups of patients were provided with implant-stabilized mandibular overdentures supported by straight ovoid prefabricated bars with a resilient joint or parallel-sided bars with a rigid joint. Measurements of plaque index, mucosal cuff health and height, marginal bone height, pathology of the denture-bearing mucosa, and patient satisfaction were correlated with the different bar designs. Ovoid bars with a resilient joint between the denture and the bar have been shown to give a slightly increased incidence of problems associated with the denture-bearing mucosa. Furthermore, the only significant mean increase in recession of the mucosal cuff was found on the distal surfaces of the distal abutments in this group of patients.

The design and synthesis of purine inhibitors of CDK2. III.

Cyclin-dependent kinases (CDKs) belong to a class of enzymes that control the ability of a cell to enter into and proceed through the cell division cycle. Using purine as a scaffold, we have synthesized a number of nanomolar inhibitors of CDK-2/cyclin E. In this report, the synthesis of a series of piperidine-substituted purine analogs will be presented, as well as some of their in vitro and in vivo biological effects.

Constipation and diarrhea: the neglected symptoms.

OBJECTIVE: To review the symptom experience of constipation and diarrhea related to cancer and its treatment. DATA SOURCES: Published articles and book chapters relating to constipation and diarrhea in patients with cancer. CONCLUSIONS: Constipation and diarrhea often represent a major concern and source of discomfort for the cancer patients. Research is needed to establish prevention and treatment protocols for patients at risk for constipation or diarrhea. IMPLICATIONS FOR NURSING PRACTICE: Oncology nurses are in an excellent position to recognize individuals who are at high risk for constipation and diarrhea. Preventive strategies and treatment protocols are of utmost importance.