Infections by Renibacterium salmoninarum and Nanophyetus salmincola Chapin are associated with reduced growth of juvenile Chinook salmon, Oncorhynchus tshawytscha (Walbaum), in the Northeast Pacific Ocean.

We examined 1454 juvenile Chinook salmon, Oncorhynchus tshawytscha (Walbaum), captured in nearshore waters off the coasts of Washington and Oregon (USA) from 1999 to 2004 for infection by Renibacterium salmoninarum, Nanophyetus salmincola Chapin and skin metacercariae. The prevalence and intensities for each of these infections were established for both yearling and subyearling Chinook salmon. Two metrics of salmon growth, weight residuals and plasma levels of insulin-like growth factor-1, were determined for salmon infected with these pathogens/parasites, both individually and in combination, with uninfected fish used for comparison. Yearling Chinook salmon infected with R. salmoninarum had significantly reduced weight residuals. Chinook salmon infected with skin metacercariae alone did not have significantly reduced growth metrics. Dual infections were not associated with significantly more severe effects on the growth metrics than single infections; the number of triple infections was very low and precluded statistical comparison. Overall, these data suggest that infections by these organisms can be associated with reduced juvenile Chinook salmon growth. Because growth in the first year at sea has been linked to survival for some stocks of Chinook salmon, the infections may therefore play a role in regulating these populations in the Northeast Pacific Ocean.

Differential growth in estuarine and freshwater habitats indicated by plasma IGF1 concentrations and otolith chemistry in Dolly Varden Salvelinus malma.

This study employed a combination of otolith microchemistry to indicate the recent habitat use, and plasma concentrations of the hormone insulin-like growth factor 1 (IGF1) as an index of recent growth rate, to demonstrate differences in growth and habitat use by Dolly Varden Salvelinus malma occupying both freshwater and estuarine habitats in south-west Alaska. Extensive sampling in all habitats revealed that fish had higher IGF1 levels in estuarine compared to lake habitats throughout the summer, and that the growth rates in different habitats within the estuary varied seasonally. In addition, otolith microchemistry indicated differentiation in estuarine habitat use among individual S. malma throughout summer months. Although growth in the estuary was higher than in fresh water in nearly all sites and months, the benefits and use of the estuarine habitats varied on finer spatial scales. Therefore, this study further illustrates the diverse life histories of S. malma and indicates an evaluation of the benefits of marine waters needs to include sub-estuary scale habitat use.

Growth and endocrine effects of recombinant bovine growth hormone treatment in non-transgenic and growth hormone transgenic coho salmon.

To examine the relative growth, endocrine, and gene expression effects of growth hormone (GH) transgenesis vs. GH protein treatment, wild-type non-transgenic and GH transgenic coho salmon were treated with a sustained-release formulation of recombinant bovine GH (bGH; Posilac). Fish size, specific growth rate (SGR), and condition factor (CF) were monitored for 14 weeks, after which endocrine parameters were measured. Transgenic fish had much higher growth, SGR and CF than non-transgenic fish, and bGH injection significantly increased weight and SGR in non-transgenic but not transgenic fish. Plasma salmon GH concentrations decreased with bGH treatment in non-transgenic but not in transgenic fish where levels were similar to controls. Higher GH mRNA levels were detected in transgenic muscle and liver but no differences were observed in GH receptor (GHR) mRNA levels. In non-transgenic pituitary, GH and GHR mRNA levels per mg pituitary decreased with bGH dose to levels seen in transgenic salmon. Plasma IGF-I was elevated with bGH dose only in non-transgenic fish, while transgenic fish maintained an elevated level of IGF-I with or without bGH treatment. A similar trend was seen for liver IGF-I mRNA levels. Thus, bGH treatment increased fish growth and influenced feedback on endocrine parameters in non-transgenic but not in transgenic fish. A lack of further growth stimulation of GH transgenic fish suggests that these fish are experiencing maximal growth stimulation via GH pathways.

Footwear and running cardio-respiratory responses.

This study compared cardio-respiratory responses during running wearing a motion control shoe (MC) or a cushioning shoe (CU) in a cross-over single blinded design. Fourteen runners (10F/4M, age=27.3+/-5.1 years, body mass=64.1+/-12.2 kg, height=167.8+/-7.5 cm, VO (2)max=52.3+/-8.8 ml/kg/min) completed a 40-min run at approximately 65% VO (2) max under both shoe conditions. Oxygen uptake (mL/kg/min; L/min), minute ventilation (L/min), respiratory exchange ratio, and heart rate were measured at minutes 8-10, 18-20, 28-30 and 38-40 of exercise. Rating of perceived exertion was obtained at minutes 10, 20, 30 and 40. Two (footwear) by four (time) repeated measures ANOVAs showed no differences between footwear conditions in overall oxygen consumption (MC=36.8+/-1.5 vs. CU=35.3+/-1.4 mL/kg/min, p=0.143), minute ventilation (MC=50.4+/-4 vs. CU=48.5+/-3.8, p=0.147), respiratory exchange ratio (MC=0.90+/-0.01 vs. CU=0.89+/-0.01, p=0.331), heart rate (MC=159+/-3 vs. CU=160+/-3, p=0.926), or rate of perceived exertion. The design of motion control footwear does not appear to affect cardio-respiratory or perceived exertion responses during submaximal running. The findings are specific to the shoes tested. Nonetheless, the outcomes suggest that footwear selection to reduce certain overuse injuries does not increase the work of running.

Endocrine effects of growth hormone overexpression in transgenic coho salmon.

Non-transgenic (wild-type) coho salmon (Oncorhynchus kisutch), growth hormone (GH) transgenic salmon (with highly elevated growth rates), and GH transgenic salmon pair fed a non-transgenic ration level (and thus growing at the non-transgenic rate) were examined for plasma hormone concentrations, and liver, muscle, hypothalamus, telencephalon, and pituitary mRNA levels. GH transgenic salmon exhibited increased plasma GH levels, and enhanced liver, muscle and hypothalamic GH mRNA levels. Insulin-like growth factor-I (IGF-I) in plasma, and growth hormone receptor (GHR) and IGF-I mRNA levels in liver and muscle, were higher in fully fed transgenic than non-transgenic fish. GHR mRNA levels in transgenic fish were unaffected by ration-restriction, whereas plasma GH was increased and plasma IGF-I and liver IGF-I mRNA were decreased to wild-type levels. These data reveal that strong nutritional modulation of IGF-I production remains even in the presence of constitutive ectopic GH expression in these transgenic fish. Liver GHR membrane protein levels were not different from controls, whereas, in muscle, GHR levels were elevated approximately 5-fold in transgenic fish. Paracrine stimulation of IGF-I by ectopic GH production in non-pituitary tissues is suggested by increased basal cartilage sulphation observed in the transgenic salmon. Levels of mRNA for growth hormone-releasing hormone (GHRH) and cholecystokinin (CCK) did not differ between groups. Despite its role in appetite stimulation, neuropeptide Y (NPY) mRNA was not found to be elevated in transgenic groups.

Studies on the mode of action of cholera toxin. Effects on solubilized adenylate cyclase.

To gain further insight into the mechanism of action of cholera toxin, solubilized preparations of adenylate cyclase from control and toxin-treated rat livers were studied. Adenylate cyclase activity was measured in both particulate and solubilized form in rat liver under control conditions and after intravenous injection of cholera toxin. Cholera toxin caused a 3.3-fold activation of adenylate cyclase in the particulate preparation and a 5.8-fold increase in the solubilized preparation. Thus, the ability of cholera toxin to stimulate adenylate cyclase is present even when the enzyme membrane environment is disrupted. Furthermore, the solubilized enzyme, after treatment with cholera toxin, retained its ability to respond to catecholamines, but not to glucagon. In contrast, the control enzyme lost its responsiveness to catecholamines and glucagon after solubilization.

Stimulation of osmotic water flow in toad bladder by prostaglandin E1. Evidence for different compartments of cyclic AMP.

The effect of prostaglandin E1 (PGE1) on osmotic water flow across toad bladder and cyclic AMP content of the mucosal epithelial cells has been determined under basal conditions and in the presence of either theophylline or antidiuretic hormone (ADH); Under basal conditions and with PGE1 concentrations from 10(-8) to 10(-5) M no evidence of stimulation of water flow was observed, and with 10(-7) M PGE1 a significant inhibition was foundmcyclic AMP content under control conditions was 8 pmol/mg protein. It was 9 at 10(-8) M PGE1, 13 at 10(-7) M, 16 at 10(-6) M, and 23 at 10(-5) M. In the presence of theophylline, 10(-8) and 10(-7) M PGE1 inhibited the theophylline-induced water flow as expected. In contrast, 10(-6) and 10(-5) M PGE1 enhanced the rate of water flow. Theophylline increased cyclic AMP content from 8 to 18 pmol/mg protein. PGE1 in the presence of theophylline caused marked increases in cyclic AMP content; The content was 23 at 10(-7) M, 41 at 10(-6) M, and 130 at 10(-5) M; Thus PGE1 stimulates theophylline-induced water flow at cyclic AMP concentrations somewhere between 23 and 41 pmol/mg. Further evidence along these lines was obtained from experiments in which the effects of PGE1 on ADH-induced water flow were studied. Inhibitory effects of PGE1 were not observed at concentrations of PGE1 which raised the level of intracellular cyclic AMP to 30 pmol/mg protein or higher. These results were obtained despite the fact that all four concentrations of PGE1 tested were found capable of inhibiting ADH-induced water flow under appropriate conditions or, in other words, were inhibiting the adenylate cyclase controlling water flow, Thus the increase in cyclic AMP content in response to PGE1 is not derived from this enzyme. Thus the stimulation of water flow by PGE1 in the presence of theophylline is thought to be caused by cyclic AMP spilling over from one compartment to the water flow compartment. No evidence was obtained to directly suggest spillover into the sodium transport compartment. Furthermore evidence is discussed to suggest that most of the cyclic AMP generated in the tissue does not originate from the enzyme controlling sodium transport. As cyclic AMP-stimulated water flow and sodium transport are thought to occur in one cell type, the granular cells, distinct pools of cyclic AMP are thought to be present in one and the same cell type. Thus one pool controls water flow and one controls sodium transport. With high concentrations of PGE1 in the presence of theophylline or high concentrations of ADH, the adenylate cyclase responsible for water flow is inhibited; However, PGE1 can stimulate a tissue adenylate cyclase to sufficiently high levels that cyclic AMP spills over into the "water flow compartment" and thus stimulates water flow.

Differences in susceptibility to tumor necrosis factor alpha-induced apoptosis among MCF-7 breast cancer cell variants.

Widespread use of MCF-7 human breast carcinoma cells as a model system for breast cancer has led to variations in these cells between different laboratories. Although several reports have addressed these differences in terms of proliferation and estrogenic response, variations in sensitivity to apoptosis have not yet been described. Tumor necrosis factor alpha (TNF-alpha) has been shown to both induce apoptosis and inhibit proliferation in MCF-7 cells. We observed that TNF-alpha inhibited proliferation in MCF-7 cell variants from three different laboratories (designated M, L, and N). MCF-7 M cells were resistant to TNF-alpha-induced apoptosis, whereas MCF-7 L cells were moderately resistant to the effect of TNF-alpha. A third variant, MCF-7 N, underwent apoptosis when exposed to TNF-alpha. Analysis of the p55 TNF-alpha receptor (TNFR) 1 expression revealed the greatest expression in MCF-7 N cells, whereas the MCF-7 L and M cells expressed 89 and 67% of MCF-7 N cell TNFR1 levels, respectively. Ceramide generation occurred in all three variants in response to TNF-alpha treatment, with MCF-7 N cells expressing the greatest increase. Cleavage of the CPP32/caspase 3 substrate poly(ADP-ribose) was observed in MCF-7 N and L cells as early as 3 and 6 h, respectively, but poly(ADP-ribose) cleavage was not observed in MCF-7 M cells. The delayed protease activation in the L variant may represent the mechanism by which these cells display delayed sensitivity to TNF-a-induced apoptosis. Expression of the Bcl-2, Mcl-1, Bcl-X, Bax, and Bak proteins was analyzed to determine whether the differences in MCF-7 cell sensitivity to apoptosis could be correlated to the differential expression of these proteins. Whereas Bak, Bcl-X, and Mcl-1 levels were identical between variants, the levels of Bcl-2 were 3.5-3.8-fold higher and the levels of Bax were 1.5-1.7-fold lower in the resistant variants (M and L) as compared with those of the sensitive variant (N). Taken together, these results suggest that differences in susceptibility to TNF-alpha-induced apoptosis among MCF-7 breast cancer cell variants may be explained by differences in TNFR expression, ceramide generation, differential expression of the Bcl-2 family of proteins, and protease activation.

Protein kinase C alpha protein expression is necessary for sustained erythropoietin production in human hepatocellular carcinoma (Hep3B) cells exposed to hypoxia.

Although protein kinase C (PKC) has been implicated as an effector of erythropoietin (EPO) production, its exact role is still uncertain. Hep3B human hepatocellular carcinoma cells were used for this study and were depleted of PKC in three different ways: long-term treatment with phorbol 12-myristate 13-acetate (PMA), selective inhibition with calphostin C, and treatment with PKCalpha antisense oligonucleotides. When EPO-producing Hep3B cells were incubated in 1% O2 (hypoxia) for 24 h, PMA treatment resulted in significant decreases in medium levels of EPO in Hep3B cell cultures at concentrations higher than 10 nM. The specific PKC inhibitor, calphostin C, significantly inhibited medium levels of EPO and EPO mRNA levels in Hep3B cells exposed to 1% O2. Western blot analysis revealed that Hep3B cells express the classical PKCalpha and gamma isoforms, as well as novel PKCepsilon and delta and the atypical zeta isoform. Preincubation with PMA for 6 h specifically down-regulated PKCalpha protein expression. Phosphorothioate modified antisense oligonucleotides specific for PKCalpha also decreased EPO production in Hep3B cells exposed to hypoxia for 20 h when compared to PKCalpha sense treatment. The translocation of PKCalpha from the soluble to particulate fractions was increased in Hep3B cells incubated under hypoxia compared with normoxia (21% O2) controls. These results suggest that the PKCalpha isoform plays an important role in sustaining hypoxia-regulated EPO production.

Nuclear protein kinase C activity is decreased in Friend erythroleukemia cells induced to differentiate.

Although it is well known that protein kinase C (PKC) is an important signaling molecule in Friend erythroleukemia cells it is not clear what role PKC may play in either regulated or unregulated erythroid cell proliferation and differentiation. The purpose of this study was to test the hypothesis that a decrease in nuclear PKC activity is associated with the induction of differentiation in Friend erythroleukemia cells. The effects of staurosporine, a selective inhibitor of PKC, and the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate, an activator of PKC, on Friend cell proliferation and differentiation were examined. Neither the inhibitor nor the activator of PKC affected proliferation at 96 h as measured by [3H]thymidine incorporation, but both compounds inhibited cell differentiation. In addition, nuclear PKC activity was highest in untreated and in tumor promoter-treated cells that were not differentiated, and it was lowest in cells induced to differentiate with hexamethylene bisacetamide or dimethylsulfoxide. It is concluded that nuclear PKC activity is essential for Friend erythroleukemia cell proliferation, and that a decrease in enzyme activity within the nucleus is associated with differentiation.

Possible involvement of phospholipase activation in erythroid progenitor cell proliferation.

The possible role of phospholipase activation in erythroid progenitor (colony-forming units erythroid; CFU-E) cell proliferation was investigated using a variety of inhibitors of phospholipase activity. Quinacrine and alpha-p-dibromoacetophenone (BPB), both nonselective phospholipase inhibitors, were tested for their effects on CFU-E-derived colony formation. Both drugs significantly inhibited colony formation in the presence or absence of added erythropoietin (Epo). For quinacrine, the concentration causing 50% inhibition (IC50) was 1 microM, whereas for BPB the IC50 was 25 microM. A more selective inhibitor of phospholipase A2, 7,7-dimethyleicosadienoic acid (DEDA), was also tested and the IC50 was 250 microM in the presence of Epo. The addition of phospholipase A or C did not significantly affect CFU-E colony formation, although arachidonic acid increased CFU-E formation as did 12-hydroperoxyeicosatetraenoic acid (12-HPETE), whereas 12-hydroxyeicosatetraenoic acid (12-HETE), 15-HETE, and 15-HPETE had no effect. These findings suggest an important role for phospholipase activation in erythroid progenitor cell proliferation.

The neurotrophins and their receptors: structure, function, and neuropathology.

The neurotrophins are a family of polypeptides that promote differentiation and survival of select peripheral and central neurons. Nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and neurotrophin-5 are included in this group. In recent years, tremendous advances have been made in the study of these factors. This has stimulated our review of the field, characterizing the neurotrophins from initial isolation to molecular analysis. The review also discusses their synthesis, localization, and responsive tissues, in both the periphery and CNS. The complex receptor interactions of the neurotrophins are also analyzed, as are putative signal transduction mechanisms. Discussion of the observed and postulated involvement in neuropathological disorders leads to the conclusion that the neurotrophins are involved in the function and dysfunction of the nervous system.

Decreased erythroid colony-forming cell response of XTfm/Y mice to testosterone and 5 beta-dihydrotestosterone.

Although certain tissue of XTfm/Y mice are known to be deficient in androgen-binding sites, the defect has not been documented for bone marrow. To study the bone marrow response to testosterone and its metabolites, we compared the responses of the erythroid colony-forming cell (CFU-E) in these mice and their wild counter-parts, XTa/Y mice, using an in vitro methyl cellulose system in the presence of erythropoietin. Testosterone or 5 beta-dihydrotestosterone (5 beta-DHT) treatment (5 mg/kg) of Ta/Y mice in vivo before femoral bone marrow culture resulted in a significant increase in CFU-E colonies. However, similar in vivo treatment of Tfm/Y mice with testosterone or 5 beta-DHT had no effect on CFU-E colony formation. 5 alpha-DHT had no significant effect on Ta/Y or Tfm/Y mouse bone marrow. Testosterone or 5 beta-DHT added directly to Ta/Y marrow cultures caused an enhancement of CFU-E colony numbers compared with erythropoietin alone. 5 alpha-DHT inhibited colony formation at high concentrations. Testosterone had no effect on Tfm/Y erythroid colonies, whereas 5 beta-DHT and 5 alpha-DHT significantly inhibited colony formation. These results indicate that Tfm/Y erythroid bone marrow colonies may have an altered response to testosterone and its metabolites compared to that of Ta/Y erythroid colonies. We postulate that the pattern of androgen receptors in the erythroid progenitor cell compartment of Tfm/Y mice is different from that of Ta/Y mice.

Interleukin-3 or erythropoietin induced nuclear localization of protein kinase C beta isoforms in hematopoietic target cells.

Protein kinase C (PKC) has been implicated in the signal transduction pathways for the biological effect of both interleukin-3 (IL-3) and erythropoietin (EPO) in hematopoietic target cells. The goal of this study was to identify specific classical isoforms of PKC and their localization in hematopoietic cells in response to the growth factors, IL-3 or EPO. In addition to murine fetal liver cells as a source of normal erythroid progenitor cells, we have utilized the B6SUt.EP cell line, a non-transformed hematopoietic cell line that requires IL-3 for proliferation, but for which EPO can substitute as a growth factor. With polyclonal antibodies prepared against peptide sequences specific for the alpha, beta I, beta II and gamma isoforms of PKC, we have identified beta I and beta II as the predominant nuclear isoforms in target cells that proliferate in response to IL-3 or EPO.

Changes in redox affect the activity of erythropoietin RNA binding protein.

We have previously identified a cytosolic protein, erythropoietin RNA binding protein (ERBP), which is up-regulated in certain tissues in response to hypoxia. To further characterize the interaction of ERBP and erythropoietin (EPO) mRNA, we have examined the role of reduction-oxidation in the EPO mRNA binding mechanism of ERBP isolated from human hepatoma cells (Hep3B). Reducing agents dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) increased ERBP binding activity in a concentration-dependent manner, whereas the oxidizing agent, diamide, abolished ERBP binding activity. In addition, treatment of Hep3B cell lysates with the irreversible sulfhydryl alkylating agent N-ethylmaleimide resulted in inhibition of the EPO mRNA-ERBP complex. Taken together, these findings suggest that sulfhydryl groups may play a role in vivo in the regulation of EPO production through the modulation of ERBP binding activity.

Inhibitory effects of zidovudine in erythroid progenitor cells. Reversal with a combination of erythropoietin and interleukin-3.

To investigate the mechanisms that may be involved in zidovudine (AZT)-induced hematopoietic toxicity, spleen cells isolated from phenylhydrazine-treated anemic mice or murine bone marrow erythroid progenitor cells were treated with AZT (1-10 microM) for 24 hr. A concentration-dependent inhibition of the binding of 125I-labeled erythropoietin (Epo) was observed, suggesting down-regulation of Epo receptors. To determine if this effect is due to modulation of the levels of Epo receptor mRNA and to assess the effect of AZT on the expression of protooncogenes, mRNA levels were monitored by the slot blot hybridization technique. AZT caused a concentration-dependent inhibition in the levels of the mRNA of Epo receptors and c-fos, whereas the level of c-myc mRNA was unaffected. AZT also inhibited protein kinase C (PKC) activity in a concentration- and time-dependent manner, causing 50% inhibition at 10 microM within 3 hr. Simultaneous addition of Epo or interleukin-3 (IL-3) partially reversed the inhibitory effects of AZT on the levels of the mRNAs and on PKC activity; however, a combination of Epo and IL-3 was significantly more effective. These studies demonstrate that (i) AZT-induced down-regulation of Epo receptors and c-fos expression coupled with inhibition of Epo receptor-mediated signal transduction through PKC are significant contributory factors to AZT-induced erythroid toxicity, and (ii) these inhibitory effects can be overcome by treatment with a combination of Epo and IL-3.

Coordinated effects of electromagnetic field exposure on erythropoietin-induced activities of phosphatidylinositol-phospholipase C and phosphatidylinositol 3-kinase.

Initial studies with the erythropoietin-sensitive human hematopoietic cell line, TF1, demonstrated both multifarious effects of pulsed electromagnetic field (EMF) exposure on lipid signal transduction and antiproliferative effects of EMF. Stimulation of TF1 cells with erythropoietin resulted in increased phosphatidylinositol 3-kinase activity within 2 min. Addition of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, produced a decrease in cell proliferation as measured by accumulation of cells in the G0/G1 phase of the cell cycle and suppression of erythropoietin-induced DNA synthesis. Similar effects on cell proliferation were seen under EMF treatment. Phosphatidylinositol 3-kinase activity in erythropoietin-stimulated TF1 cells, measured in whole-cell extracts, increased 34% within 2 min and remained above basal levels for at least 20 min. EMF decreased erythropoietin-stimulated phosphatidylinositol 3-kinase activity to lower than basal levels. Additionally, translocation of the 85-kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase to the membrane was prevented by EMF. Phosphatidylinositol-specific phospholipase C was activated, as reflected by increases in diacylglycerol and inositol trisphosphate at 15-60 s after EMF treatment. These results provide the first evidence of subtle coordinated changes by EMF associated with loss of phosphatidylinositol 3-kinase activity, inhibition of the translocation of p85 to the membrane, and activation of phosphatidylinositol-phospholipase C.

Identification of environmental chemicals with estrogenic activity using a combination of in vitro assays.

Environmental chemicals that function as estrogens have been suggested to be associated with an increase in disease and dysfunctions in animals and humans. To characterize chemicals that may act as estrogens in humans, we have compared three in vitro assays which measure aspects of human estrogen receptor (hER)-mediated estrogenicity. Chemicals were first tested for estrogen-associated transcriptional activity in the yeast estrogen screen (YES). This was created by expressing hER and two estrogen response elements linked to the lacZ gene in yeast. Second, chemicals that were tested in YES were then assayed for direct interaction with hER in a competition binding assay. Third, chemicals were tested in the estrogen-responsive MCF-7 human breast cancer cell line transiently transfected with a plasmid containing two estrogen response elements linked to the luciferase gene. Together, these assays have identified two metabolites of DDT, o,p'-DDD and p,p'-DDD, that have estrogenic activity. Interestingly, previous studies had reported that the DDD metabolites were nonestrogenic in whole animal models. Alachlor, the most frequently used herbicide in the United States, cis-nonachlor, and trans-nonachlor displayed weak estrogenic activity in the combined assays. The antifungal agent benomyl had no estrogenic activity. We propose that a combination of in vitro assays can be used in conjunction with whole animal models for a more complete characterization of chemicals with estrogenic activity.

Dexniguldipine hydrochloride inhibits growth of human pancreatic adenocarcinoma cells and expression of protein kinase C isoforms alpha and zeta.

New therapeutic regimens are needed for the treatment of pancreatic cancer. In view of the importance of protein kinase C (PKC) in tumorigenesis, we evaluated the effects of dexniguldipine hydrochloride (DNIG) on the in vitro growth of CFPAC-1 human pancreatic adenocarcinoma cells and the expression of PKC isoforms. DNIG is a potent antineoplastic drug with well-established anti-PKC activity and the ability to reverse multidrug resistance. DNIG (1.6-25 mu M) decreased the number of cells in culture in a time- and dose-dependent manner with an EC(50) of 4.9 mu M on day 1 and 2.8 mu M on day 3. When PKC isoform expression in CFPAC-1 cells was analyzed by immunoblotting, the predominant isoforms were identified as alpha and zeta. The expression of PKC alpha and zeta was inhibited significantly by DNIG (0.63-2.5 mu M). These results suggest that DNIG suppresses the proliferation of CFPAC-1 pancreatic cancer cells, possibly by inhibiting the expression of specific PKC isoforms.

Dexniguldipine hydrochloride inhibits growth of human ht-29 colon-carcinoma cells and expression of protein-kinase-C-delta and protein-kinase-C-zeta.

Previous studies have implicated protein kinase C (PKC) in colon carcinogenesis, but a clear understanding of the role of PKC in colon cancer is lacking. The purpose of this study was to investigate the effects of dexniguldipine hydrochloride (DNIG) on the in vitro growth of HT-29 human colon carcinoma cells and the expression of PKC isoforms. DNIG is a selective inhibitor of PKC that binds specifically to the regulatory region and is also a potent antineoplastic drug with an ability to reverse multidrug resistance. DNIG (1.6-25 mu M) decreased the number of HT-29 cells in culture in a time- and dose-dependent manner with an EC(50) of 1.4 mu M on day 3. Predominant PKC isoforms expressed in HT-29 cells were identified as Delta and zeta by immunoblotting. The expression of PKC Delta and zeta was inhibited significantly by DNIG (0.16-1.25 mu M). These results suggest that the suppression of the growth of HT-29 colon carcinoma cells by DNIG involves the inhibition of the expression of PKC Delta and zeta.