Prevention of diabetes-induced abnormal retinal blood flow by treatment with d-alpha-tocopherol

Hyperglycemia in diabetes mellitus has been shown to activate diacylglycerol (DAG)-protein kinase C (PKC) pathway in the vascular tissues, possibly altering vascular function. We have characterized the effects of vitamin E (d-alpha-tocopherol) on activation of PKC and DAG levels in retinal tissues of diabetic rats, and correlated its effects to retinal hemodynamics using video-based fluorescein angiography (VFA). Comparing streptozotocin-induced diabetic rats to controls, membranous PKC specific activities were increased by 71% (p < 0.05). Western blot analysis showed that the membranous PKC beta II isoform was significantly increased by 133 +/- 45% (p < 0.05). Intraperitoneal injection of d-alpha-tocopherol (40 mg/kg) every other day prevented the increases in membranous PKC specific activity and PKC beta II protein shown by immunoblots. Similar to PKC activities, total DAG levels were increased in the retina and were normalized by d-alpha-tocopherol treatment. Physiologically, abnormalities of retinal blood hemodynamics, as measured using VFA, which previously have been reported to be associated with increases of DAG and PKC levels in the diabetic rats, were prevented by d-alpha-tocopherol treatment in diabetic rats. The direct effect of d-alpha-tocopherol on total DAG and [3H]-palmitate incorporation into DAG were also examined using cultured bovine retinal endothelial cells (REC). Exposure of REC to 22 mM glucose for three days increased total DAG and [3H]-palmitate labeled DAG levels by 35 +/- 8% and 50 +/- 8%, respectively (p < 0.05). The presence of d-alpha-tocopherol (50 micrograms/ml) prevented the increase of both total DAG and [3H]-palmitate labeled DAG levels in cells exposed to 22 mM glucose. These findings suggested that the mechanism of the d-alpha-tocopherol's effect appears to be mediated by the normalization of the hyperglycemia-induced activation of the DAG-PKC pathway which leads to the normalization of abnormal retinal blood flow seen in diabetes mellitus.     

Shape changes and chemokinesis of Walker carcinosarcoma cells: effects of protein kinase inhibitors (HA-1004, polymyxin B, sangivamycin and tamoxifen) and an inhibitor of diacylglycerol kinase (R 59022)

Previous work has shown that PMA and diacylglycerols, activators of protein kinase C (PKC) can suppress cell polarity and locomotor activity of Walker carcinosarcoma cells in vitro, suggesting that PKC activation may result in a stop signal for tumor cell locomotion. This hypothesis was further analysed. The present results show that the DAG kinase inhibitor, R 59022, suppressed tumor cell polarity and strongly inhibited cell locomotion at a concentration of 10(-4), thus supporting the earlier finding that an increased availability of DAGs can suppress the locomotor activity of Walker carcinosarcoma cells. The results support the stop-signal hypothesis of PKC activation insofar as DAG kinase inhibition mimics the effects of DAGs and PMA. In order to clarify further the effects of protein kinase modulation on locomotion, we now extended our studies on structurally different inhibitors of protein kinases. In contrast to H-7, HA-1004 had no effect on cell polarity and did not reduce cell locomotion in the presence of colchicine, but reduced the proportion of spontaneously locomoting cells by 70% at 3 x 10(-4) M. Polymyxin B suppressed cell polarity and locomotion only at concentrations that proved to be toxic. Tamoxifen had no significant effect on cell polarity and locomotor activity. Sangivamycin did not suppress cell polarity and spontaneous locomotion at a concentration range of 10(-9) M to 10(-4) M. However, at 10(-4) M it decreased the proportion of migrating, colchicine-stimulated cells by 50%. The diverse responses to structurally different PKC inhibitors may be explained by their limited and variable specificity for PKC and different mechanisms of action on PKC.     

Activation of protein kinase C by tumor necrosis factor-alpha in human non-pigmented ciliary epithelium

Previously, we have shown that tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, increases the synthesis and release of endothelin-1 (ET-1), a potent vasoactive peptide from human non-pigmented ciliary epithelial (HNPE) cells, in a protein kinase C (PKC)-dependent manner. Diacylglycerol (DAG) and intracellular calcium ([Ca2+]i) are well known activators of PKC. Some cytokines induce PKC activation by stimulating phospholipase C that hydrolyzes phosphatidylinositol bisphosphate (PIP2) into IP3 (intracellular calcium mobilizer) and DAG. In this study, the existence of a similar pathway was evaluated in HNPE cells treated with TNF-alpha, using intracellular calcium ([Ca2+]i) measurements, PKC translocation assays and thin-layer chromatography (TLC) for quantification of DAG. Incubation times for agonists and inhibitors ranged from 1-30 minutes. The increase in DAG levels with TNF-alpha treatment was consistent with the observed translocation of the calcium-dependent PKC alpha isoform from the cytosol to the plasma membrane. However, these observations were not accompanied by a concomitant increase in [Ca2+]i. Similar translocation responses were observed with phorbol ester (phorbol 12-myristate 13-acetate) treatment. Our results indicate that TNF-alpha-induced PKC activation in HNPE cells occurs as a result of elevated DAG levels and is not due to an increase in intracellular calcium. Activated PKC, could enhance the pro-inflammatory responses of TNF-alpha in part by increasing the production of endothelins in the eye.     

Prevention of glomerular dysfunction in diabetic rats by treatment with d-alpha-tocopherol

Because d-alpha-tocopherol (vitamin E) has been shown to decrease diacylglycerol (DAG) levels and prevent the activation of protein kinase C (PKC), which is associated with retinal and renal dysfunctions in diabetes, the study presented here characterized the effect of d-alpha-tocopherol treatment to prevent glomerular hyperfiltration and increased albuminuria as well as PKC activities in streptozotocin (STZ)-induced diabetic rats. Two weeks after the induction of diabetes, total DAG content and PKC activity in glomeruli were significantly increased in diabetic rats by 106.4 +/- 16.8% and 66.4 +/- 8.4%, respectively, compared with control rats. Intraperitoneal injection of d-alpha-tocopherol (40 mg/kg of body weight) every other day prevented the increases in total DAG content and PKC activity in glomeruli of diabetic rats. Glomerular filtration rate (GFR) and filtration fraction (FF) were significantly elevated to 4.98 +/- 0.34 mL/min and 0.36 +/- 0.05, respectively, in diabetic rats, compared with 2.90 +/- 0.14 mL/min and 0.25 +/- 0.02, respectively, in control rats. These hemodynamic abnormalities in diabetic rats were normalized to 2.98 +/- 0.09 mL/min and 0.24 +/- 0.01, respectively, by d-alpha-tocopherol. Albuminuria in 10-wk diabetic rats was significantly increased to 9.1 +/- 2.2 mg/day compared with 1.2 +/- 0.3 mg/day in control rats, whereas d-alpha-tocopherol treatment improved albumin excretion rate to 2.4 +/- 0.6 mg/day in diabetic rats. To clarify the mechanism of d-alpha-tocopherol's effect on DAG-PKC pathway, the activity and protein levels of DAG kinase alpha and gamma, which metabolize DAG to phosphatidic acid, were examined. Treatment with d-alpha-tocopherol increased DAG kinase activity in the glomeruli of both control and diabetic rats, by 22.6 +/- 3.6% and 28.5 +/- 2.3% respectively, although no differences were observed in the basal DAG kinase activity between control and diabetic rats. Because immunoblotting studies did not exhibit any difference in the protein levels of DAG kinase alpha and gamma, the effect of d-alpha-tocopherol is probably modulating the enzyme kinetics of DAG kinase. These findings suggest that the increases in DAG-PKC pathway play an important role for the development of glomerular hyperfiltration and increased albuminuria in diabetes and that d-alpha-tocopherol treatment could be preventing early changes of diabetic renal dysfunctions by normalizing the increases in DAG and PKC levels in glomerular cells.     

Production of second messengers following chemotactic and mitogenic urokinase-receptor interaction in human fibroblasts and mouse fibroblasts transfected with human urokinase receptor

We studied urokinase-type plasminogen activator (u-PA)-dependent chemotaxis and DNA synthesis in both human fibroblasts and LB6 mouse fibroblasts transfected with human u-PA receptor (u-PAR) gene (LB6 clone 19). Both cell lines have receptors for the amino-terminal fragment of u-PA (u-PA-ATF). We observed that u-PA and u-PA-ATF stimulated chemotactic migration of both LB6 clone 19 cells and human fibroblasts, which could be impaired by down-regulation of protein kinase C (PKC) with phorbol myristate acetate (PMA). While LB6 clone 19 cells were unable to undergo mitosis following exposure to either u-PA or u-PA-ATF, human fibroblasts were stimulated to mitosis by exogenous addition of native u-PA, and u-PA-ATF was ineffective. The mitogenic activity of u-PA on human fibroblasts could also be impaired by down-regulation of PKC with PMA. We studied second messenger formation following u-PAR stimulation. Neither inositol lipid metabolism nor intracellular Ca2+ content were affected, while an increase of diacylglycerol (DAG) generation was observed. Such DAG formation was related to de novo synthesis from glucose and was dependent on ligand-receptor interaction. Both u-PA-ATF and the native u-PA molecule were able to stimulate DAG formation, u-PA being from three to fourfold more efficient than ATF. These data suggest that u-PAR stimulation per se is sufficient to trigger DAG formation. The native molecule confers on the cell an additional stimulus, possibly related with the activation of a u-PA-catalytic site-dependent substrate. Such stimulation allows the cell to reach the DAG threshold level required to trigger DNA synthesis.     

p53-independent apoptosis induced by paclitaxel through an indirect mechanism

The efficacy of chemotherapeutic agents may be determined by a number of different factors, including the genotype of the tumor cell. The p53 tumor suppressor gene frequently is mutated in human tumors, and this may contribute to chemotherapeutic resistance. We tested the requirement for wild-type p53 in the response of tumor cells to treatment with paclitaxel (trade name Taxol), an antineoplastic agent that stabilizes cellular microtubules. Although paclitaxel is broadly effective against human tumor xenografts in mice, including some known to carry p53 mutations, we found that p53-containing mouse tumor cells were significantly more sensitive to direct treatment with this drug than were p53-deficient tumor cells. In an attempt to reconcile this apparent discrepancy, we examined the requirement for p53 in the cytotoxic effects of tumor necrosis factor alpha (TNF-alpha), a cytokine released from murine macrophages upon paclitaxel treatment. Conditioned medium from paclitaxel-treated macrophages was capable of inducing p53-independent apoptosis when applied to transformed mouse embryonic fibroblasts and was inhibitable by antibodies against TNF-alpha. Furthermore, in response to direct treatment with TNF-alpha, both wild-type and p53-deficient tumor cells underwent apoptosis to similar extents and with similar kinetics. Our results suggest that the efficacy of paclitaxel in vivo may be due not only to its microtubule-stabilizing activity, but its ability to activate local release of an apoptosis-inducing cytokine.     

Erythropoietin stimulates nuclear localization of diacylglycerol and protein kinase C beta II in B6SUt.EP cells

Erythropoietin (EPO) is a hormone, as well as a hematopoietic growth factor, that specifically regulates the proliferation and differentiation of erythroid progenitor cells. Although the membrane-bound receptor for EPO has no intrinsic kinase activity, it triggers the activation of protein kinases via phospholipases A2, C, and D. A cascade of serine and threonine kinases, including Raf-1, MAP kinase and protein kinase C (PKC) is activated following tyrosine phosphorylation. In this study, we have examined whether changes in nuclear PKC and 1,2-diacylglycerol (DAG) are induced following EPO treatment of the murine target cell line, B6SUt.EP. Western blot analysis using isoform-specific antibodies demonstrated the presence of PKC beta II, but not PKC alpha, beta I, gamma, epsilon, delta, eta, or zeta in the nuclei of cells stimulated with EPO. The increase in nuclear beta II levels was accompanied by an immediate rise in DAG mass levels with both of the increases peaking by 1 min. These rapid increases in nuclear DAG and PKC beta II expression suggest a mechanism for EPO-induced changes in gene expression necessary for cell proliferation.     

p53 mutation and chemoresistance in oral-maxillofacial squamous cell carcinoma. Role of p53 in the cell cycle control and in the modulating action of chemotherapeutic agents

p53 is a "tumor suppressor gene" with a basic function in the cellular cycle control and subsequently in the induction of the neoplastic process. p53 found changed in the majority of malignant human tumors. In the oral and maxillofacial cancers p53 mutations varies from 4% to 60% of the cases. Researches in vitro in tumors of the colon-rectum, breast, lung, ovary, testicle, bladder and in leukaemia, show a correlation between p53 overexpression (mutation) and resistance to the anti-tumor agents. The functional connection between the p53 and the chemotherapic drugs action which cause a direct DNA damage, is the apoptosis, a physiologic mechanism activated by p53 for regulating cell growth but also indispensable for the cytotoxic effects (apoptosis is an irreversible process culminating in cell death). Loss of the p53 activity (mutation) in the tumoral cells determines non-activation of the apoptosis and the drug resistance. These results have led to early clinical applications. In the breast cancers the p53 is already utilized as chemoresistance marker, directing the therapy, if changed to alternative drugs (Taxolo) which have a p53-independent action. Even into cell lung cancers a retroviral vector containing the wild-type p53 gene was produced to mediate transfer of wild-type p53, noting tumor regression. In oral and maxillofacial tumors surgery is elective. It is emphasized that, in advanced cancers, it is included in multimode protocols where the neoadjuvant chemotherapy has an important clinical function, with precise indications. The possibility to determine previously the p53 "status" in the cancer cells by genetic study, may give a specific factor of screening, indicative of the tumor chemoresponse, together with other well-known prognostic factors, with advantage for the therapeutic programming and especially for the known surgical treatment.     

Experimental immunization with anti-rheumatic bacterial extract OM-89 induces T cell responses to heat shock protein (hsp)60 and hsp70; modulation of peripheral immunological tolerance as its possible mode of action in the treatment of rheumatoid arthritis (RA)

High intracellular 1,2,-sn-diacylglycerol (DAG) usually activates protein kinase C (PKC). In choline-deficient Fischer 344 rats, we previously showed that fatty liver was associated with elevated hepatic DAG and sustained activation of PKC. Steatosis is a sequelae of many liver toxins, and we wanted to determine whether fatty liver is always associated with accumulation of DAG with activation of PKC. Obese Zucker rats had 11-fold more triacylglycerol in their livers and 2-fold more DAG in their hepatic plasma membrane than did lean control Zucker rats. However, this increased diacylglycerol was not associated with translocation or activation of PKC in hepatic plasma membrane (activity in obese rats was 897 pmol/mg protein X min(-1) vs. 780 pmol/mg protein X min(-1) in lean rats). No differences in PKC isoform expression were detected between obese and lean rats. In additional studies, we found that choline deficiency in the Zucker rat did not result in activation of PKC in liver, unlike our earlier observations in the choline deficient Fischer rat. This dissociation between fatty liver, DAG accumulation and PKC activation in Zucker rats supports previous reports of abnormalities in PKC signaling in this strain of rats.     

Immunodominant minor histocompatibility antigen peptides recognized by cytolytic T lymphocytes primed by indirect presentation

We have previously shown that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in growth zone chondrocyte (GC) differentiation and that this effect is mediated by protein kinase C (PKC). The aim of the present study was to identify the signal transduction pathway used by 1,25(OH)2D3 to stimulate PKC activation. Confluent, fourth passage GC cells from costochondral cartilage were used to evaluate the mechanism of PKC activation. Treatment of GC cultures with 1,25(OH)2D3 elicited a dose-dependent increase in both inositol-1,4,5-trisphosphate and diacylglycerol (DAG) production, suggesting a role for phospholipase C and potentially for phospholipase D. Addition of dioctanoylglycerol to plasma membranes isolated from GCs increased PKC activity. Neither pertussis toxin nor choleratoxin had an inhibitory effect on PKC activity in control or 1,25(OH)2D3-treated GCs, indicating that neither Gi nor Gs proteins were involved. Phospholipase A2 inhibitors, quinacrine, OEPC (selective for secretory phospholipase A2), and AACOCF3 (selective for cytosolic phospholipase A2), and the cyclooxygenase inhibitor indomethacin decreased PKC activity, while the phospholipase A2 activators melittin and mastoparan increased PKC activity in GC cultures. Arachidonic acid and prostaglandin E2, two downstream products of phospholipase A2 action, also increased PKC activity. These results indicate that 1,25(OH)2D3-dependent stimulation of PKC activity is regulated by two distinct phospholipase-dependent mechanisms: production of DAG, primarily via phospholipase C and production of arachidonic acid via phospholipase A2.     

Reinitiation of DNA synthesis and cell division in senescent human fibroblasts by microinjection of anti-p53 antibodies

In human fibroblasts, growth arrest at the end of the normal proliferative life span (induction of senescence) is dependent on the activity of the tumor suppressor protein p53. In contrast, once senescence has been established, it is generally accepted that reinitiation of DNA synthesis requires loss of multiple suppressor pathways, for example, by expression of Simian virus 40 (SV40) large T antigen, and that even this will not induce complete cell cycle traverse. Here we have used microinjection of monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, to reinvestigate the effect of blocking p53 function in senescent human fibroblasts. Unexpectedly, we found that both antibodies induce senescent cells to reenter S phase almost as efficiently as SV40, accompanied by a reversion to the "young" morphology. Furthermore, this is followed by completion of the cell division cycle, as shown by the appearance of mitoses, and by a four- to fivefold increase in cell number 9 days after injection. Immunofluorescence analysis showed that expression of the p53-inducible cyclin/kinase inhibitor p21sdi1/WAF1 was greatly diminished by targeting p53 with either PAb1801 or DO-1 but remained high and, moreover, still p53 dependent in cells expressing SV40 T antigen. As previously observed for induction, the maintenance of fibroblast senescence therefore appears to be critically dependent on functional p53. We suggest that the previous failure to observe this by using SV40 T-antigen mutants to target p53 was most probably due to incomplete abrogation of p53 function.     

Effects of chronic alcohol use and age on human lymphocyte protein kinase C activity

The effects of alcohol exposure on human peripheral circulating lymphocyte protein kinase C (PKC) activity were characterized in lymphocytes harvested from two sample groups. The first group (control) consisted of 30 nonalcoholic male subjects and the second group consisted of nine male subjects with chronic alcoholism. Alcoholic subjects were admitted for detoxification to a substance abuse unit located in a nonprofit community hospital. In this group of subjects, blood was sampled on admission for detoxification (pre-A), and after 5 days (post-A). Subjects received chlordiazepoxide for treatment of alcohol withdrawal symptoms. PKC activities measured in the control, pre-A, and post-A groups expressed as pmol/microgram/min +/- SEM were 5.09 +/- 0.50, 1.81 +/- 0.43, and 3.95 +/- 0.44. Control PKC was significantly higher than pre-A PKC (p < or = 0.05) and post-A PKC was significantly higher than pre-A PKC (p < or = 0.05). Total lymphocyte PKC activity was also found to be inversely related to age, expressed by the relationship log(PKC) = 0.870-0.005(Age), with R = 0.433.     

Contributions to maxima in protein kinase C activation

In many lipid systems, the activity of protein kinase C (PKC) exhibits a peak followed by a decline as the mol % of one component is increased. In these systems, an increase in one lipid component is always at the expense of another or accompanied by a change in total lipid concentration. Here we report that in saturated phosphatidylserine (PS)/phosphatidylcholine (PC)/diacylglycerol (DAG) mixtures, increasing PS or DAG at the expense of PC revealed an optimal mol % PS, dependent on mol % DAG, with higher mol % PS diminishing activity. The decrease at high mol % PS is probably not attributable simply to more gel-phase lipid due to the higher melting temperature of saturated PS versus PC because a similar peak in activity occurred in unsaturated lipid systems. Increasing the total lipid concentration at suboptimal mol % PS provided the same activity as higher mol % PS at lower total lipid concentration. However, at optimal mol % PS, activity increased and then decreased as a function of total lipid concentration. PKC autophosphorylation also exhibited an optimum as a function of mol % PS, and increasing the PKC concentration increased the mol % PS at which activity decreased, both for autophosphorylation and for heterologous phosphorylation. Formation of two-dimensional crystals of PKC on lipid monolayers also exhibited a peak as a function of mol % PS, and the unit cell size of the crystals formed shifts from 50 x 50 A at low mol % PS to 75 x 75 A at higher PS. Collectively, these data suggest the existence of optimal lipid compositions for PKC activation, with increased quantity of these domains serving to dilute out enzyme-substrate aggregates and/or enzyme-enzyme aggregates on the lipid surface.     

p53-dependent ceramide response to genotoxic stress

Both p53 and ceramide have been implicated in the regulation of growth suppression. p53 has been proposed as the "guardian of the genome" and ceramide has been suggested as a "tumor suppressor lipid. " Both molecules appear to regulate cell cycle arrest, senescence, and apoptosis. In this study, we investigated the relationship between p53 and ceramide. We found that treatment of Molt-4 cells with low concentrations of actinomycin D or gamma-irradiation, which activate p53-dependent apoptosis, induces apoptosis only in cells expressing normal levels of p53. In these cells, p53 activation was followed by a dose- and time-dependent increase in endogenous ceramide levels which was not seen in cells lacking functional p53 and treated similarly. Similar results were seen in irradiated L929 cells whereby the p53-deficient clone was significantly more resistant to irradiation and exhibited no ceramide response. However, in p53-independent systems, such as growth suppression induced by TNF-alpha or serum deprivation, ceramide accumulated irrespective of the upregulation of p53, indicating that p53 regulates ceramide accumulation in only a subset of growth-suppressive pathways. Finally, ceramide did not increase p53 levels when used at growth-suppressive concentrations. Also, when cells lacking functional p53, either due to mutation or the expression of the E6 protein of human papilloma virus, were treated with exogenous ceramide, there was equal growth suppression, cell cycle arrest, and apoptosis as compared with cells expressing normal p53. These results indicate that p53 is unlikely to function "downstream" of ceramide. Instead, they suggest that, in situations where p53 performs a critical regulatory role, such as the response to genotoxic stress, it functions "upstream" of ceramide. These studies begin to define a relationship between these two pathways of growth inhibition.     

Flavopiridol induces apoptosis in chronic lymphocytic leukemia cells via activation of caspase-3 without evidence of bcl-2 modulation or dependence on functional p53

Flavopiridol has been reported to induce apoptosis in lymphoid cell lines via downregulation of bcl-2. The in vitro activity of flavopiridol against human chronic lymphocytic leukemia (CLL) cells and potential mechanisms of action for inducing cytotoxicity were studied. The in vitro viability of mononuclear cells from CLL patients (n = 11) was reduced by 50% at 4 hours, 24 hours, and 4 days at a flavopiridol concentration of 1.15 micromol/L (95% confidence interval [CI] +/-0.31), 0.18 micromol/L (95% CI +/-0.04), and 0.16 micromol/L (95% CI +/-0.04), respectively. Loss of viability in human CLL cells correlated with early induction of apoptosis. Exposure of CLL cells to 0.18 micromol/L of flavopiridol resulted in both decreased expression of p53 protein and cleavage of the caspase-3 zymogen 32-kD protein with the appearance of its 20-kD subunit. Contrasting observations of others in tumor cell lines, flavopiridol cytotoxicity in CLL cells did not correlate with changes in bcl-2 protein expression alterations. We evaluated flavopiridol's dependence on intact p53 by exposing splenocytes from wild-type (p53(+/+)) and p53 null (p53(-/-)) mice that demonstrated no preferential cytotoxicity as compared with a marked differential with F-ara-a and radiation. Incubation of CLL cells with antiapoptotic cytokine interleukin-4 (IL-4) did not alter the LC50 of flavopiridol, as compared with a marked elevation noted with F-ara-a in the majority of patients tested. These data demonstrate that flavopiridol has significant in vitro activity against human CLL cells through activation of caspase-3, which appears to occur independently of bcl-2 modulation, the presence of IL-4, or p53 status. Such findings strongly support the early introduction of flavopiridol into clinical trials for patients with B-CLL.     

Gene therapy for primary and metastatic pancreatic cancer with intraperitoneal retroviral vector bearing the wild-type p53 gene

BACKGROUND: Metastatic pancreatic cancer is uniformly fatal because no effective chemotherapy is available. Mutations in the p53 tumor suppressor gene are found in up to 70% of pancreatic adenocarcinomas. We examined the efficacy of a retroviral vector containing the wild-type p53 gene on metastatic pancreatic cancer in a nude mouse model. METHODS: Bxpc3 human pancreatic cancer cells were transduced with either a retroviral p53 vector or an LXSN empty vector. Cells were examined for incorporation of tritiated thymidine to determine the effect of p53 retroviral transduction on DNA synthesis, and a TACS2 assay for apoptosis was performed. The functional activity of p53 in transduced cells was assessed by Western blot analysis with an antibody to WAF1/p21. In vivo effects of intraperitoneal injections of the p53 vector were examined in a nude mouse model of peritoneal carcinomatosis. RESULTS: Cells treated with the p53 vector exhibited a 59% to 85.5% reduction in cell number compared with the control cells (P < .05). p53-treated cells demonstrated decreased incorporation of tritiated thymidine (12.7% +/- 0.7% vs 17.5% +/- 1.4%; P = .002), increased staining for apoptosis, and increased expression of the WAF1/p21 protein. Treatment of nude mice with the retroviral p53 vector resulted in a significant inhibition of growth of the primary pancreatic tumor, as well as the peritoneal tumor deposits, compared with the LXSN control vector. CONCLUSIONS: Intraperitoneal delivery of a retroviral p53 vector may provide a novel treatment approach for peritoneal carcinomatosis from pancreatic cancer.     

Interaction of urokinase-type plasminogen activator with its receptor rapidly induces activation of glucose transporters

The interaction of urokinase-type plasminogen activator (u-PA) or of u-PA amino-terminal fragment (u-PA-ATF) with the cell surface receptor (u-PAR) was found to stimulate an increase of glucose uptake in many cell lines, ranging from normal and transformed human fibroblasts, mouse fibroblasts transfected with human u-PAR, and cells of epidermal origin. Such increase of glucose uptake reached a peak within 5-10 min, depending on the cell line, and occurred through the facilitative glucose transporters (GLUTs), since it was inhibited by cytochalasin B. Each cell line showed a specific mosaic of glucose transporter isoforms, GLUT2 being the most widespread and GLUT1 the most abundant, when present. u-PAR stimulation was followed by translocation of GLUT1 from the microsomal to the membrane compartment, as shown by both immunoblotting and immunofluorescence of sonicated plasma membrane sheets and by activation of GLUT2 on the cell surface. Both translocation and activation resulted inhibitable by protein-tyrosine kinase inhibitors and independent of downregulation of protein kinase C (PKC). The increase of intracellular glucose was followed by neosynthesis of diacylglycerol (DAG) from glucose, as previously shown. Such neosynthesis was completely inhibited by impairment of facilitative GLUT transport by cytochalasin B. DAG neosynthesis was followed by activation of PKC, whose activity translocated into the intracellular compartment (PKM), where it probably phosphorylates substrates required for u-PAR-dependent chemotaxis. Our data show that u-PAR-mediated signal transduction, related with u-PA-induced chemotaxis, involves activation of tyrosine kinase-dependent glucose transporters, leading to increased de novo DAG synthesis from glucose, eventually resulting in activation of PKC.