Enhanced protein C activation and inhibition of fibrinogen cleavage by a thrombin modulator.

A modulator of the enzymatic activity of human thrombin, designated LY254603, was identified that enhances the thrombin-catalyzed generation of the anticoagulant factor activated protein C, yet inhibits thrombin-dependent fibrinogen clotting. By means of mutant substrates, it was shown that LY254603 mediates the change in enzymatic substrate specificity through an alteration in thrombin's S3 substrate recognition site, a mechanism that appeared to be independent of allosteric changes induced by either sodium ions or by thrombomodulin. This compound may represent the prototype of a class of agents that specifically modulates the balance between thrombin's procoagulant and anticoagulant functions.

Negative regulation of the human polyomavirus BK enhancer involves cell-specific interaction with a nuclear repressor.

We have examined the cell type-specific regulation of the human BK virus (BKV) enhancer. This enhancer functions efficiently in cis to activate expression from the adenovirus major late promoter in the human kidney cell line, 293, and in a monkey kidney cell line, MK2, but not in the HeLa cell line. In gel retardation migration assays, specific BKV enhancer-protein complexes could be observed by using nuclear extracts prepared from each cell line. Moreover, a unique DNA-protein complex was observed by using the HeLa cell nuclear extracts. By DNase footprint analysis, four binding regions for HeLa cell nuclear proteins were defined within the BKV enhancer repeat region. Two of the protected regions encompassed nuclear factor 1 or CCAAT transcription factor binding sites. These nuclear factor 1 sites also were protected by nuclear proteins from the 293 and MK2 cell lines. The other two protected sites encompassed a region of symmetry which included a sequence similar to the simian virus 40 TC enhancer motif and to a conserved sequence present upstream or within the introns of several cellular genes. These two sites were not protected by either the 293 or MK2 nuclear proteins. Competition studies in transfected cells indicated that the reduced activity of the BKV enhancer in the HeLa cell line was due to negative regulation. Further, we have demonstrated that binding of a nuclear factor(s) to the HeLa cell-specific site is involved in the repression of enhancer activity.

Activation of the adenovirus and BK virus late promoters: effects of the BK virus enhancer and trans-acting viral early proteins.

We have examined the activation of the adenovirus major late promoter (MLP) by the cis-acting enhancer element of the human polyomavirus BK and by the trans-acting simian virus 40 (SV40) T antigen and adenovirus E1A proteins. By using chloramphenicol acetyltransferase expression vectors, we found that the MLP (pLP-CAT) was trans-activated in human and monkey kidney cells expressing the SV40 T antigen. In addition, the MLP could be cis-activated by the BK virus enhancer in both human and monkey kidney cells; approximately 20 times more chloramphenicol acetyltransferase was produced from expression vectors containing a hybrid promoter (BL), in which the BK enhancer was upstream of the MLP, than from expression vectors containing the MLP alone. This same level of enhancement of the MLP by the BK enhancer was observed in cells expressing the T antigen of SV40. However, in the 293 cell line, greater enhancement of MLP activity (70-fold) was observed with the BK enhancer sequence. In contrast, MLP activity in the 293 cell line was unchanged by the SV40 enhancer. In cotransfection experiments, MLP activity, augmented by the BK enhancer, could be further stimulated with a plasmid coding for the E1A gene products. By creating deletion mutants, we determined that the high-level activation of the hybrid BL transcriptional unit by the E1A proteins requires both MLP sequences and an intact BK virus enhancer. On the other hand, activation of the BL transcriptional unit by the T antigen did not require an intact enhancer sequence. Our results suggest that the SV40 T antigen and E1A proteins trans-activate the BL promoter by different mechanisms. We also demonstrate in cotransfection experiments that the BK late promoter is activated 45-fold by the SV40 T antigen.

E1A-induced enhancer activity of the poly(dG-dT).poly(dA-dC) element (GT element) and interactions with a GT-specific nuclear factor.

The alternating sequence poly(dG-dT).poly(dA-dC) is a highly repeated sequence in the eucaryotic genome. We have examined the effect of trans-acting early viral proteins on the ability of the GT element to stimulate transcription of the adenovirus major late promoter (MLP). We find that the GT element alone does not activate expression from the MLP in either the presence or absence of another enhancer element. However, in the presence of the E1A gene products of either adenovirus type 5 or 2, the GT element activated expression from the MLP. The stimulatory activity of the GT element in the presence of E1A had the properties of an enhancer element, and the trans-activating effect on the GT element was additive in conjunction with the E1A-responsive BK virus enhancer. We also have demonstrated that a specific nuclear factor(s) binds to the GT element. However, the E1A protein(s) do not affect the initial factor interaction(s) with the GT element. Overall, our data demonstrate that trans modulation of promoter activity can be mediated through the GT element.

Frequency of prolonged remission duration after high-dose cytarabine intensification in acute myeloid leukemia varies by cytogenetic subtype.

Advances in the treatment of acute myeloid leukemia (AML) have occurred with the introduction of new therapies including high-dose cytarabine and the identification of powerful prognostic factors such as cytogenetics that predict for long-term outcome. To date, the prognostic impact of cytarabine dose escalation within various cytogenetic groups of AML has not been assessed. We describe 285 newly diagnosed patients with primary AML who had adequate karyotypes and were enrolled on a prospective Cancer and Leukemia Group B cytogenetic study. All patients were randomly assigned to postremission treatment with standard-, intermediate-, or high-dose cytarabine intensification. Patients were categorized to one of three cytogenetic groups: (a) core binding factor type [(CBF); ie., t(8;21) inv(16), t(16;16), and del(16)]; (b) normal; and (c) other abnormality karyotype. An evaluation of these patients after a median follow-up time of over 7 years was performed to determine the relationship of intensification to outcome by cytogenetic group. Patients included 57 patients with CBF AML, 140 patients with normal karyotype AML, and 88 patients with other cytogenetic abnormalities. The treatment outcome of CBF AML patients was superior, with an estimated 50% still in complete remission (CR) after 5 years as compared with 32 and 15% for patients with normal karyotype AML and other abnormality AML, respectively (P < 0.001). Univariate analysis showed the following nonkaryotype factors to predict a prolonged CR duration: (a) younger age (P < 0.008); (b) lower leukocyte count (P=0.01); (c) the presence of Auer rods (P=0.004); (d) a lower percentage of bone marrow blasts (P=0.001) at the time of diagnosis, (e) and a higher postremission cytarabine dose (P < 0.001). The impact of cytarabine dose on long-term remission was most marked (P < 0.001) in the CBF AML group (after 5 years, 78% of those with a dose of 3 g/m2 were still in CR, 57% of those with a dose of 400 mg/m2 were still in CR, and 16% of those with a dose of 100 mg/m2 were still in CR) followed by normal karyotype AML (P=0.01; after 5 years, 40% of those with a dose of 3 g/m2 were still in CR, 37% of those with a dose of 400 mg/m2 were still in CR, and 20% of those with a dose of 100 mg/m2 were still in CR). In contrast, cytarabine at all doses produced only a 21% or less chance of long-term continuous CR for patients with other cytogenetic abnormalities. A multivariate analysis of CR duration assessed the independent impact of each of these variables on cure. Significant factors entering this model in descending order of importance were cytogenetic group (CBF > normal > other abnormality; P=0.00001), cytarabine dose (3 g/m2 > 400 mg/m2 > 100 mg/m2; P=0.00001), logarithm of leukocyte count at the time of diagnosis (P=0.0005), and histological subtype of AML (P=0.005). This study demonstrates that the curative impact of cytarabine intensification varies significantly among cytogenetic groups and results in a substantial prolongation of CR among patients with CBF and normal karyotypes, but not in those with other karyotypic abnormalities. These findings support the use of pretreatment cytogenetics in risk stratification of postremission AML therapy.

Risk factors for high-dose cytarabine neurotoxicity: an analysis of a cancer and leukemia group B trial in patients with acute myeloid leukemia.

PURPOSE: We analyzed pretreatment characteristics of patients with postremission acute myeloid leukemia (AML) treated with high-dose cytarabine (HIDAC) during a recent Cancer and Leukemia Group B (CALGB) trial to determine risk factors associated with HIDAC neurotoxicity. PATIENTS AND METHODS: One hundred seventy-six patients received at least one course of HIDAC as part of a CALGB protocol designed to determine the optimal dose of cytarabine (ara-C) for postremission treatment of AML. HIDAC consisted of 3 g/m2 ara-C infused over 3 hours at 12-hour intervals on days 1, 3, and 5. The pretreatment characteristics of 170 patients were available for risk analyses. RESULTS: Eighteen patients (10%) experienced neurotoxicity. Univariate analyses demonstrated associations between the occurrence of neurotoxicity and elevated serum creatinine, age, and alkaline phosphatase (AP). Multivariate analysis showed that these variables were independent risk factors. These findings were used to construct a risk model with the following parameters: creatinine greater than or equal to 1.2 mg/dL, age greater than or equal to 40 years, and AP greater than or equal to 3 x normal. Seventeen of 46 (37%) patients with two or more of these criteria developed neurotoxicity compared with one of 124 (1%) patients with one or none. The sensitivity and specificity of this model were 94% and 81%, respectively. CONCLUSION: We conclude that patients with two or more of the following parameters may be at increased risk for HIDAC neurotoxicity: (creatinine greater than or equal to 1.2 mg/dL, age greater than or equal to 40, and AP greater than or equal to 3 x normal). However, this model should be confirmed by analysis of additional groups of patients treated with HIDAC.

Differential central effects of mineralocorticoid and glucocorticoid agonists and antagonists on blood pressure.

Systolic blood pressure was measured, using an indirect tail method, in conscious male rats at several time intervals after the intracerebroventricular injection of mineralo-and glucocorticoid agonists and antagonists. Intracerebroventricular administration of the antimineralocorticoid RU 28318 (10 ng) decreased blood pressure, while the antiglucocorticoid RU 38486 (10 ng) caused an increase, which was slower in onset and of longer duration. The effect of the antimineralocorticoid was maximal at 8 h and had disappeared after 24 h. The antiglucocorticoid had a significant effect 24 and 48 h after injection. Neither antagonist was effective when administered sc at the same dose (10 ng). Intracerebroventricular administration of aldosterone (10 ng) and the selective glucocorticoid agonist RU 28362 (10 ng) increased and decreased blood pressure, respectively. Corticosterone given intracerebroventricularly (10-100 ng) did not affect blood pressure unless the dose was increased to 1 microgram. Two weeks after adrenalectomy a decrease in blood pressure was observed when the rats were given 0.9% saline instead of water to drink. Replacement therapy with corticosterone (12.5-mg steroid pellet, sc) restored blood pressure to the level in the sham-operated controls. The chronically elevated level of circulating corticosterone produced by a 100-mg sc corticosterone pellet increased blood pressure. The 12.5-and 100-mg sc corticosterone pellets resulted in plasma corticosterone levels of approximately 3 and 20 micrograms/100 ml, respectively. Intracerebroventricular administration of the glucocorticoid and mineralocorticoid antagonists (10 ng) increased and decreased, respectively, the blood pressure of the adrenalectomized rats receiving corticosterone substitution. From these data we conclude that corticosteroids can affect the central regulation of blood pressure. The mineralo- and glucocorticoids have opposite effects, which differ in onset and duration. The mineralocorticoids increased blood pressure, whereas the glucocorticoid decreased it.

17beta-estradiol, but not raloxifene, decreases thrombomodulin in the antithrombotic protein C pathway.

Raloxifene is a nonsteroidal selective estrogen receptor modulator (SERM) that mimics the effects of estrogen on some plasma lipids and may have direct effects on the vascular wall. The objective of this study was to determine the effects of 17beta-estradiol, raloxifene, and LY139,478 (a related benzothiophene SERM) on the anticoagulant protein C pathway. In human vascular endothelial cells activated with interleukin-1 (IL-1), we demonstrated decreased thrombomodulin-dependent protein C activation. 17beta-estradiol reduced the anticoagulant properties of both unstimulated and IL-1-activated endothelial cells by decreasing thrombomodulin expression. In contrast, raloxifene and LY139,478 enhanced the anticoagulant properties of both unstimulated and IL-1-activated endothelial cells through upregulation of thrombomodulin. Regulation of the protein C pathway via thrombomodulin on vascular endothelium may be a novel mechanism by which SERMs could potentially confer cardioprotective effects and reduce the thrombotic risk associated with HRT in compromised patients.

Amplification of multicistronic plasmids in the human 293 cell line and secretion of correctly processed recombinant human protein C.

We have constructed multicistronic vectors containing the cDNAs for murine dihydrofolate reductase (DHFR), hygromycin phosphotransferase (HyPR), and human protein C (HPC), an antithrombotic factor. Using a sequential selection protocol with hygromycin (Hy) and methotrexate (MTX), we demonstrate the selective amplification of the murine dhfr cDNA in the adenovirus-transformed human kidney cell line 293, and the coamplification of the cDNA for HPC. Such recombinant 293 cell lines secreted HPC at levels as high as 25 micrograms/10(6) cells/day. In addition, we found that the complex vitamin K-dependent posttranslational modification of gamma-carboxylation of glutamate was not limiting at these high secretion levels, although the proteolytic processing of the protein was slightly reduced. Further, the HPC secreted from the gene-amplified cell lines had full anticoagulant activity when compared to plasma-derived HPC.

High-level expression of secreted proteins from cells adapted to serum-free suspension culture.

We have developed a host cell/vector system based on the use of adenovirus-transformed cells and a promoter, designated GBMT, capable of being activated by the Ela tumor antigen produced in these cells. GBMT-based vectors were constructed with hygromycin phosphotransferase and murine dihydrofolate reductase as selective markers. We demonstrate their utility in two adenovirus-transformed cell lines, human kidney 293 and Syrian hamster AV12-664. Further, we describe methods and conditions for the direct adaptation of isolated recombinant clones to serum-free suspension growth conditions. For exemplary purposes, we describe the generation of stable recombinant 293 cell lines with single-copy integrated vectors secreting the highly complex clotting factor human protein C at levels as high as 20 mg/l in serum-free suspension culture. In addition, using the AV12-664 cell line with GBMT and direct dominant selection of the dhfr gene, we have isolated clones secreting a tissue plasminogen activator derivative at levels of about 40 mg/l under serum-free suspension conditions. The distinct advantages of this vector/host cell system are 1) the direct selection of stable clones expressing relatively high levels of recombinant protein, eliminating the need for the tedious stepwise gene amplification process and 2) the direct adaptation to serum-free suspension culture.

Differential mechanisms of plasminogen activator inhibitor-1 gene activation by transforming growth factor-beta and tumor necrosis factor-alpha in endothelial cells.

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (SERPIN) specific for tissue-type and urokinase-like plasminogen activators. High plasma PAI-1 activity is a risk factor for thrombotic diseases. Due to the short half-life of PAI-1, regulation of PAI-1 gene expression and secretion of active PAI-1 into the blood stream is important for hemostatic balance. We have investigated transcriptional control of PAI-1 gene expression in bovine aortic endothelial cells (BAECs) and human cell lines using PAI-1 5' promoter-luciferase reporter assays. Contrary to the cytokine-induced up-regulation of PAI-1 mRNA and protein levels, we found that only transforming growth factor-beta (TGF-beta) was efficient in inducing PAI-1 promoter activation. Tissue necrosis factor-alpha (TNF-alpha) induced a small luciferase activity with the 2.5 kb PAI-1 promoter, but not with the PAI-800/4G/5G and p3TP-lux promoters. Next we investigated whether a lack of response to TNF-alpha was due to deficient signaling pathways. BAECs responded to TNF-alpha with robust NFkappaB promoter activation. TGF-beta activated the p38 MAP kinase, while TNF-alpha activated both the SAPK/JNK and p38 MAP kinases. The ERK1/2 MAP kinases were constitutively activated in BAECs. BAEC therefore responded to TNF-alpha stimulation with activation of the MAP kinases and the NFkappaB transcriptional factors. We further measured the messenger RNA stability under the influence by TGF-beta and TNF-alpha and found no difference. PAI-1 gene activation by TNF-alpha apparently is yet to be defined for the location of the response element and/or the signaling pathway, while TGF-beta is the most important cytokine for PAI-1 transcriptional activation through its 5' proximal promoter.

Brain mineralocorticoid receptor diversity: functional implications.

Mineralocorticoid receptors (MRs) in neurons of the anterior hypothalamus and the periventricular brain regions mediate aldosterone-selective actions on sodium homeostasis, salt appetite and cardiovascular regulation. Corticosterone is not effective in these neurons, possibly because it is enzymatically inactivated. However, MRs in limbic brain regions, notably in the hippocampal neurons, do already respond to very low concentrations of both corticosterone and aldosterone. The MR-mediated effects stabilize neuronal transmission and appear critical for neuronal integrity of a sub-region of the hippocampus: the dentate gyrus. Higher concentrations of corticosterone induced by stress and the circadian rise progressively activate the lower affinity glucocorticoid receptors (GRs), which in coordination with MR-mediated actions then facilitate adaptive processes required for recovery of homeostasis. It is postulated that this balanced MR- and GR-mediated action of corticosterone is of critical importance for regulation of the stress response and behavioural adaptation.

Activation of glucocorticoid receptors and the effect of naloxone during hemorrhagic hypotension.

Evidence indicates that endogenous opioid peptides and glucocorticoids participate in the control of cardiovascular regulation during hemorrhagic shock. In the present study, we investigated a possible interaction between brain opioid peptides and adrenal corticosteroids regarding the control of arterial pressure during hemorrhage. The bleeding volumes required to lower arterial pressure to 80, 60 and 40 mmHg were studied in anesthetized sham-operated (SHAM) and adrenalectomized (ADX) rats. I.c.v. administration of 10 micrograms of naloxone resulted in a significant increase in the bleeding volume required to lower arterial pressure from 60 to 40 mmHg in SHAM animals, whereas no effect of naloxone was observed in ADX animals. Replacement therapy with a 100% corticosterone pellet (100 mg, s.c.), but not with a 12.5% corticosterone pellet (12.5 mg corticosterone and 87.5 mg cholesterol, s.c.), resulted in an effect of naloxone on the bleeding volume in ADX animals. The effect of replacement therapy could be inhibited by i.c.v. pretreatment with the synthetic glucocorticoid receptor antagonist, RU38486 (100 ng). These data suggest that (1) opioid mechanisms are involved in the regulation of blood pressure during hemorrhage, and (2) occupancy of glucocorticoid receptors is required for naloxone to exert its hemodynamic effect during hemorrhagic hypotension in ADX rats.

New chemotherapy treatment options and implications for nursing care.

PURPOSE/OBJECTIVES: To review the drug profiles and nursing implications of Camptosar (irinotecan) (pharmacia & Upjohn, Inc., Kalamazoo, MI), Hycamtin (topotecan) (SmithKline Beecham Oncology,Philadelphia, PA), 9-aminocamptothecin TM (9-AC) (Pharmacia & Upjohn, Inc.), Taxotere (docetaxel) (Rhône-Poulenc Rorer Pharmaceuticals, Inc., Collegeville, PA), and Oncaspar (Rhône-Poulenc Rorer Pharmaceuticals). DATA SOURCES: Published articles, abstracts, drug manufacturers, personal experience with clinical trials of these agents. DATA SYNTHESIS: Camptosar's dose-limiting toxicity is diarrhea, which requires astute management. Hycamtin and 9-AC are severely myelosuppressive. Taxotere's use has been complicated by fluid retention and, to a lesser degree, hypersensitivity reactions. Oncaspar has the same toxicity profile as natural L-asparaginase with fewer hypersensitivity reactions. CONCLUSIONS: Research in the field of oncology continues to progress toward the cure of cancer. Novel agents offer promising new treatment options and affect response rates and patient care. Oncology nurses are challenged to stay abreast of these new developments. IMPLICATIONS FOR NURSING PRACTICE: Nurses play a vital role in the assessment and management of treatment-related side effects. Patient education regarding anticipated side effects and appropriate self-care measures are essential nursing functions.

Therapeutic options for treating advanced colorectal cancer.

Despite recent decreases in overall incidence and mortality, colorectal cancer is a major health concern affecting 1 of every 18 Americans. Although potentially curable if detected early, 25% of patients present with metastatic disease, whereas another 10%-60% develop metastases resulting from the spread of microscopic disease not noted at the time of initial surgery. Historical treatment options for advanced colorectal cancer have been unsatisfactory, with survival rates of approximately 9%. This article reviews the newer treatment options that are available to patients while providing nurses with information they need to confidently care for patients receiving these treatments.

Brain corticosteroid receptors and regulation of arterial blood pressure.

A single intracerebroventricular injection of 10 ng of the mineralocorticoid aldosterone in normotensive male Wistar rats induced a transient increase in blood pressure. Corticosterone produced this response only at a 100-fold higher dose. In contrast, a decrease in systolic blood pressure occurred following the intracerebroventricular administration of a selective glucocorticoid agonist (Ru 28362; 10 ng). The intracerebroventricular administration in normotensive and adrenalectomized, corticosterone-replaced, male rats of an antimineralocorticoid (Ru 28318; 10 ng) decreased systolic blood pressure, while an antiglucocorticoid (Ru 38486; 10 ng) caused an increase. These data suggest that adrenal steroids affect central mechanisms underlying cardiovascular regulation by binding to central mineralo- and glucocorticoid receptors.

Characterization and novel purification of recombinant human protein C from three mammalian cell lines.

Human Protein C (HPC), an antithrombotic factor with potential clinical utility, is a vitamin K-dependent protein that has several complex post-translational modifications. In an effort to define the functional roles of these modifications, recombinant HPC (rHPC) was expressed in and characterized from 3 adenovirus-transformed cell lines. The rHPC in crude culture medium from the 3 cell lines displayed anticoagulant activities that were either higher, slightly lower or much lower than that of plasma HPC. The rHPC from each cell line was purified and characterized using a novel, but simple chromatographic method, termed "pseudo-affinity", capable of resolving molecules differing by only very slight modifications. We demonstrate the critical dependence of full gamma-carboxylation on the function of this protein. In addition, our data indicate that both the gamma-carboxyglutamate and glycosyl contents affect the functional activities of rHPC.