Interspecific and Spatial Comparisons of Perfluorinated Compounds in Bighead and Silver Carp in the Illinois River, Illinois, USA.

We examined perfluoroalkyl compounds (PFC) in bighead (BHCP; Hypophthalmichthys nobilis) and silver (SVCP; H. molitrix) carp from the Illinois River, Illinois, USA. Summed PFC concentrations in whole fish did not differ by species or river reach. Perfluorooctanesulfonate (PFOS) concentrations were much greater in whole fish (16.4 ng/g) than in fillets (3.4 ng/g). PFOS concentrations represented 35%-51% of total measured PFC concentrations in whole fish, and in fillets were weakly associated with carcass mass (R2=0.17, p=0.01) and % carcass lipid (R2=0.16, p=0.01). No such relationship was observed in whole fish. The relationship between concentrations of individual PFC congeners in whole fish and carcass mass or % lipid content varied by species. Our study demonstrated that filter-feeders such as BHCP and SVCP can accumulate measureable concentrations of PFC and these results are important for understanding the fate of these compounds in large river systems.

The multiple functions of heme oxygenase-1 in the liver.

Heme oxygenases (HO) are essential enzymes which degrade heme into carbon monoxide (CO), biliverdin and free iron. Due to its anti-inflammatory, anti-apoptotic and, as recently described, anti-viral properties the inducible HO isoform HO-1 is an important molecule which could find its way into therapy of gastrointestinal diseases. Acute and chronic liver injuries including acute liver failure, alcoholic or viral hepatitis, chronic inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma are life threatening diseases and as a consequence might result in the necessity of liver transplantation. HO-1 as well as its reaction products of heme degradation has been linked to cytoprotection. HO-1 induction in rodent models of acute and chronic hepatic inflammation resulted in improvement of liver damage and down-regulation of pro-inflammatory cytokine levels. Furthermore HO-1 induction interfered with fibrosis progression in mice and partially resolved existing fibrosis. Likewise, HO-1 induction interfered with replication of hepatitis viruses B and C, which frequently are the reason for chronic hepatitis and subsequent tumor growth. Liver transplantation is limited by ischemia/reperfusion (I/R) injury, which is characterized by hypoxia and nutrient deficiency resulting in oxidative stress, apoptosis and immune activation. Induction of HO-1 and application predominantly of CO have been shown to interfere with I/R liver injury and to improve recipient and graft survival. On the other hand HO-1 has been shown to be over-expressed in various tumors, including hepatocellular carcinoma (HCC). Due to its anti-apoptotic properties this bears the risk to promote tumor growth. Anti-apoptotic effects are predominantly mediated by CO. This review aims to summarize beneficial as well as detrimental effects of HO-1 and its products within the liver.

A novel technique for selective NF-kappaB inhibition in Kupffer cells: contrary effects in fulminant hepatitis and ischaemia-reperfusion.

BACKGROUND AND AIMS: The transcription factor nuclear factor kappa B (NF-kappaB) has risen as a promising target for anti-inflammatory therapeutics. In the liver, however, NF-kappaB inhibition mediates both damaging and protective effects. The outcome is deemed to depend on the liver cell type addressed. Recent gene knock-out studies focused on the role of NF-kappaB in hepatocytes, whereas the role of NF-kappaB in Kupffer cells has not yet been investigated in vivo. Here we present a novel approach, which may be suitable for clinical application, to selectively target NF-kappaB in Kupffer cells and analyse the effects in experimental models of liver injury. METHODS: NF-kappaB inhibiting decoy oligodeoxynucleotides were loaded upon gelatin nanoparticles (D-NPs) and their in vivo distribution was determined by confocal microscopy. Liver damage, NF-kappaB activity, cytokine levels and apoptotic protein expression were evaluated after lipopolysaccharide (LPS), d-galactosamine (GalN)/LPS, or concanavalin A (ConA) challenge and partial warm ischaemia and subsequent reperfusion, respectively. RESULTS: D-NPs were selectively taken up by Kupffer cells and inhibited NF-kappaB activation. Inhibition of NF-kappaB in Kupffer cells improved survival and reduced liver injury after GalN/LPS as well as after ConA challenge. While anti-apoptotic protein expression in liver tissue was not reduced, pro-apoptotic players such as cJun N-terminal kinase (JNK) were inhibited. In contrast, selective inhibition of NF-kappaB augmented reperfusion injury. CONCLUSIONS: NF-kappaB inhibiting decoy oligodeoxynucleotide-loaded gelatin nanoparticles is a novel tool to selectively inhibit NF-kappaB activation in Kupffer cells in vivo. Thus, liver injury can be reduced in experimental fulminant hepatitis, but increased at ischaemia-reperfusion.

Targeted drug delivery by in vivo coupling to endogenous albumin: an albumin-binding prodrug of methotrexate (MTX) is better than MTX in the treatment of murine collagen-induced arthritis.

OBJECTIVE: To examine the effect of an albumin-binding prodrug of methotrexate (MTX) in the treatment of murine collagen-induced arthritis (CIA). METHODS: The prodrug AWO54 with the formula EMC-d-Ala-Phe-Lys-Lys-MTX binds selectively to the cysteine-34 position of endogenous albumin, which acts as a macromolecular drug carrier for MTX to the site of inflammation. The CIA model was used to evaluate the anti-arthritic effect of the compound after intravenous application. RESULTS: The albumin-bound form of AWO54 was efficiently cleaved by cathepsin B and plasmin, two proteases that are overexpressed in rheumatoid arthritis, and release a MTX lysine derivative. AWO54 suppressed CIA in a dose-dependent manner and was significantly better than MTX. To obtain a similar effect only about 20% of the MTX-equivalent dose of AWO54 had to be given. The efficacy of the drug was tested in two different stages of CIA: while both, MTX and AWO54 inhibited arthritis in an early stage of the disease, in a later stage only AWO54 showed a significant inhibitory effect in comparison with control. CONCLUSION: Targeted drug delivery by in vivo coupling of a prodrug of MTX to endogenous albumin is better than MTX in the treatment of CIA.

TNF tolerance and cytotoxicity in the liver: the role of interleukin-1beta, inducible nitric oxide-synthase and heme oxygenase-1 in D-galactosamine-sensitized mice.

OBJECTIVE AND DESIGN: Pretreatment with tumor necrosis factor (TNF)-alpha induces tolerance towards itself in experimental liver injury. MATERIAL AND TREATMENT: To study mechanisms of TNF tolerance we used knockout mice for either TNF-receptor-2 (TNFR-2), inducible nitric oxide (NO)-synthase (iNOS) or caspase-1 (ICE) or inhibited heme oxygenase-1 (HO-1) by treatment with zinc-protoporphyrin 9. Liver damage was induced by administration of TNF to mice sensitized with D-galactosamine (GalN). Tolerance was induced by pretreatment with low doses of TNF. METHODS: Severity of liver injury was assessed by determination of plasma transaminases and apoptosis. Time courses of intra-hepatic iNOS, interleukin-1beta (IL-1beta) and HO-1 expression after TNF treatment were measured by reverse transcription polymerase chain reaction (RT-PCR). TNF-receptor-1 (TNFR-1) expression was determined by immunofluorescent staining. RESULTS: TNF-pretreatment did not affect TNFR-1 expression in the liver and resulted in time dependent up-regulation of iNOS, IL-1beta and HO-1. TNF- pretreated TNFR-2, iNOS or ICE knockout mice were as sensitive towards GalN/TNF as the wild type, while mice with impaired HO-1 activity were even more sensitive, but tolerance was inducible in all TNF-pretreated mice. CONCLUSIONS: TNF tolerance towards GalN/TNF treatment is mediated by TNFR-1. IL-1beta, iNOS and HO-1 neither mediated TNF-tolerance nor TNF cytotoxicity.

Treosulfan in the treatment of advanced ovarian cancer: a randomised co-operative multicentre phase III-study.

BACKGROUND: In August 1988 a randomised phase III multicenter trial was started in order to compare cisplatinum/treosulfan (PT) with standard cisplatinum/cyclophosphamide (PC) in advanced ovarian carcinoma, aiming at lower toxicity and maintained efficiency. PATIENTS AND METHODS: Five hundred and nineteen patients were enrolled into the protocol. Final evaluation after a median observation time of more than five years was made in July 1996 and included 398 eligible patients, of whom 366 were evaluable regarding efficiency and 290 in respect of toxicity. The tumour stages were classified as FIGO II in 53, FIGO III in 244 and FIGO IV in 68 patients. The patients were stratified regarding post-operative tumour burden. RESULTS: Hematological and gastrointestinal toxicity WHO > = 3 were comparable between the two study arms though a significant difference could be demonstrated regarding alopecia (PT 8% vs. PC 47% after six cycles). The median time to progression as the main efficiency item was in favour of the study schedule (PT 20.6 vs. PC 15.1 months) while significant differences were neither observed in the whole study group nor in the analysed subgroups (R0, < 2 cm, > = 2 cm). The same held true for overall survival. CONCLUSION: PT may be recommended as a less toxic substitute for the former standard PC. After the acceptance of paclitaxel/cisplatin as a new standard, the role of treosulfan should be investigated regarding adjuvant therapy in patients without residual tumor, as a potential partner in triple or sequential treatment and in second-line treatment.

Chronic inflammation and protection from acute hepatitis in transgenic mice expressing TNF in endothelial cells.

Endothelial activation is an important feature of many inflammatory diseases and has been implicated as the cause of vascular complications in disorders such as diabetes, atherosclerosis, and transplant rejection. One of the most potent activators of the endothelium is TNF, which can also be expressed by endothelial cells, causing a permanent, autocrine stimulatory signal. To establish a model of continuous endothelial activation and to elucidate the role of endothelial derived TNF in vivo, we generated transgenic mice expressing a noncleavable transmembrane form of TNF under the control of the endothelial-specific tie2 promoter. Adult tie2-transmembrane TNF-transgenic mice developed chronic inflammatory pathology in kidney and liver, characterized by perivascular infiltration of mononuclear cells into these organs. Along with the infiltrate, an up-regulation of the adhesion molecules ICAM-1 and VCAM-1, but not E-selectin, in the endothelium was observed. Despite predisposition to chronic inflammation these mice were protected from immune-mediated liver injury in a model of Con A-induced acute hepatitis. Although the blood levels of soluble TNF and IFN-gamma were increased in transgenic animals after challenge with Con A, no damage of hepatocytes could be detected, as assessed by the lack of increase in plasma transaminase activities and the absence of TUNEL staining in the liver. We conclude that expression of transmembrane TNF in the endothelium causes continuous endothelial activation, leading to both proinflammatory and protective events.

Low-molecular-weight hyaluronic acid induces nuclear factor-kappaB-dependent resistance against tumor necrosis factor alpha-mediated liver injury in mice.

Liver resident NK1.1+ T cells are supposed to play a pivotal role in the onset of inflammatory liver injury in experimental mouse models such as concanavalin A (Con A)-induced hepatitis. These cells, expressing the adhesion receptor, CD44, are largely depleted from the liver by a single intravenous injection of low-molecular-weight fragments of hyaluronic acid (LMW-HA). Here, we report that LMW-HA pretreatment protected mice from liver injury in several models of T-cell- and macrophage-dependent, tumor necrosis factor alpha (TNF-alpha)-mediated inflammatory liver injury, i.e., from liver injury induced by either Con A or Pseudomonas exotoxin A (PEA) or PEA/lipopolysaccharide (LPS). Interestingly, apart from inhibition of cellular adhesion, pretreatment of mice with LMW-HA was also capable of preventing hepatocellular apoptosis and activation of caspase-3 induced by direct administration of recombinant murine (rmu) TNF-alpha to D-galactosamine (GalN)-sensitized mice. LMW-HA-induced hepatoprotection could be neutralized by pretreatment with the nuclear factor-kappaB (NF-kappaB) inhibitor, pyrrolidine dithiocarbamate (PDTC), demonstrating the involvement of NF-kappaB in the observed protective mechanism. Indeed, injection of LMW-HA rapidly induced the production of TNF-alpha by Kupffer cells and the translocation of NF-kappaB into hepatocellular nuclei. Both LMW-HA-induced TNF-alpha production and NF-kappaB translocation were blocked by pretreatment with PDTC. Our findings provide evidence for an unknown mechanism of LMW-HA-dependent protection from inflammatory liver disease, i.e., induction of TNF-alpha- and NF-kappaB-dependent cytoprotective proteins within the target parenchymal liver cells.

Dissection of the intracellular pathways in hepatocytes suggests a role for Jun kinase and IFN regulatory factor-1 in Con A-induced liver failure.

Con A administration results in dose-dependent immune-mediated liver injury. Cytokines are important to determine the outcome of liver failure in this model, and especially TNF-alpha and IFN-gamma directly contribute to hepatocyte damage. The intracellular pathways of these two cytokines, which eventually result in tissue destruction, are not well defined. Here we used anti-IFN-gamma Abs and adenoviral vectors that express molecules inhibiting distinct TNF-alpha-dependent pathways in hepatocytes to better understand the relevance of specific intracellular signaling cascades for Con A-induced liver failure. We show that activation of TNF-alpha- and IFN-gamma-dependent intracellular pathways occurs prior to the influx of immune-activated cells into the liver and that anti-TNF-alpha and anti-IFN-gamma neutralizing Abs cannot block infiltration of these cells. Blocking experiments with Abs and adenoviral vectors showed that NF-kappaB activation and the Fas-associated death domain protein/caspase 8 cascade in hepatocytes during Con A-induced liver failure have no impact on tissue injury. Additionally, STAT1 activation alone after Con A injection in liver cells does not result in liver damage. In contrast, IFN-gamma-dependent expression of IFN regulatory factor-1 and TNF-alpha-dependent activation of c-Jun N-terminal kinase in liver cells correlates with liver cell damage after Con A injection. Therefore, our experiments indicate that 11418690

Inducible nitric oxide synthase is critical for immune-mediated liver injury in mice.

Concanavalin A (Con A) causes severe TNF-alpha-mediated and IFN-gamma-mediated liver injury in mice. In addition to their other functions, TNF-alpha and IFN-gamma both induce the inducible nitric oxide (NO) synthase (iNOS). Using different models of liver injury, NO was found to either mediate or prevent liver damage. To further elucidate the relevance of NO for liver damage we investigated the role of iNOS-derived NO in the Con A model. We report that iNOS mRNA was induced in livers of Con A-treated mice within 2 hours, with iNOS protein becoming detectable in hepatocytes as well as in Kupffer cells within 4 hours. iNOS-/- mice were protected from liver damage after Con A treatment, as well as in another TNF-alpha-mediated model that is inducible by LPS in D-galactosamine-sensitized (GalN-sensitized) mice. iNOS-deficient mice were not protected after direct administration of recombinant TNF-alpha to GalN-treated mice. Accordingly, pretreatment of wild-type mice with a potent and specific inhibitor of iNOS significantly reduced transaminase release after Con A or GalN/LPS, but not after GalN/TNF-alpha treatment. Furthermore, the amount of plasma TNF-alpha and of intrahepatic TNF-alpha mRNA and protein was significantly reduced in iNOS-/- mice. Our results demonstrate that iNOS-derived NO regulates proinflammatory genes in vivo, thereby contributing to inflammatory liver injury in mice by stimulation of TNF-alpha production.

TNF-alpha-induced expression of adhesion molecules in the liver is under the control of TNFR1--relevance for concanavalin A-induced hepatitis.

TNF-alpha has been clearly identified as central mediator of T cell activation-induced acute hepatic injury in mice, e.g., Con A hepatitis. In this model, liver injury depends on both TNFRs, i.e., the 55-kDa TNFR1 as well as the 75-kDa TNFR2. We show in this report that the hepatic TNFRs are not transcriptionally regulated, but are regulated by receptor shedding. TNF directly mediates hepatocellular death by activation of TNFR1 but also induces the expression of inflammatory proteins, such as cytokines and adhesion molecules. Here we provide evidence that resistance of TNFR1(-/-) and TNFR2(-/-) mice against Con A hepatitis is not due to an impaired production of the central mediators TNF and IFN-gamma. Con A injection results in a massive induction of ICAM-1, VCAM-1, and E-selectin in the liver. Lack of either one of both TNFRs did not change adhesion molecule expression in the livers of Con A-treated mice, presumably reflecting the fact that other endothelial cell-activating cytokines up-regulated adhesion molecule expression. However, treatment of TNFR1(-/-) and TNFR2(-/-) mice with murine rTNF revealed a predominant role for TNFR1 for the induction of hepatic adhesion molecule expression. Pretreatment with blocking Abs against E- and P-selectin or of ICAM(-/-) mice with anti-VCAM-1 Abs failed to prevent Con A hepatitis, although accumulation of the critical cell population, i.e., CD4(+) T cells was significantly inhibited. Hence, up-regulation of adhesion molecules during acute hepatitis unlikely contributes to organ injury but rather represents a defense mechanism.

Functional and structural defects in HIV type 1 nef genes derived from pediatric long-term survivors.

DNA sequences and three distinct in vitro functions of Nef were evaluated in a group of seven perinatally infected children. nef gene sequences obtained before and after virus culture showed that one of the five non-/slow progressors harbored a virus with large deletions. nef genes from the remaining four children were full length but contained discrete changes at a higher frequency than the rapid progressors. In functional studies, 40 of 44 Nef proteins derived from the whole study group were capable of binding the cellular serine kinase p62, indicating that this function is well conserved among naturally occurring viruses. In contrast, representative Nef proteins derived from the long-term non-/slow progressors were found to be defective or far less capable of enhancing viral replication and/or viral infectivity in herpesvirus saimiri-transformed human T cells and peripheral blood mononuclear cells. On reversion of highly prevalent point mutations in the defective proteins, viral replication could be restored to wild-type levels. Our results suggest that nef genes derived from pediatric long-term nonprogressors have gross deletions in isolated cases but a higher prevalence of discrete changes that may impair Nef function in primary T cell assays, but not all functions reported for Nef.

Pairing-dependent mislocalization of a Drosophila brown gene reporter to a heterochromatic environment.

We describe the precise positioning of a reporter gene within heterochromatin where it may be silenced. A transposition of the 59E-60A region into pericentric heterochromatin ensnares distal 59E-60A via somatic pairing. The frequency with which a brown (bw) reporter gene in 59E is silenced is influenced by chromosomal configurations. Silencing occurs only when the bw+ reporter is unpaired due to heterozygosity with a deficiency, where the frequency of bw+ reporter expression is correlated with the extent of bw gene and flanking sequence present. Surprisingly, the frequency of pairing between the transposition in heterochromatin and distal 59E observed cytologically is indistinguishable from the frequency of pairing of homologous chromosomes at 59E in wild-type larval brains, regardless of configuration. Therefore, bringing a susceptible reporter gene into close proximity with heterochromatin does not necessarily affect its expression, but local pairing changes resulting from altered chromosomal configurations can lead to silencing. We also find that an ensnared distal copy of bw that is interrupted by a heterochromatic insertion enhances silencing. This demonstrates that bw can be simultaneously acted upon by pericentric and distal blocks of heterochromatin.

Phase I clinical and pharmacokinetic study of titanocene dichloride in adults with advanced solid tumors.

This Phase I dose-escalation clinical trial of a lyophilized formulation of titanocene dichloride (MKT4) was conducted to determine the maximum tolerated dose, the dose-limiting toxicity (DLT), and pharmacokinetics of titanium (Ti) after a single i.v. infusion of MKT4. Forty patients with refractory solid malignancies were treated with a total of 78 courses. Using a modified Fibonacci scheme, 15 mg/m2 initial doses of titanocene dichloride were increased in cohorts of three patients up to level 11 (560 mg/m2) if DLT was not observed. The maximum tolerated dose was 315 mg/m2, and nephrotoxicity was DLT. Two minor responses (bladder carcinoma and non-small cell lung cancer) were observed. The pharmacokinetics of plasma Ti were assessed in 14 treatment courses by atomic absorption spectroscopy. The ratio for the area under the curve(0-infinity) in plasma and whole blood was 1.2. The following pharmacokinetic parameters were determined for plasma, as calculated in a two-compartment model: biological half-life t1/2beta in plasma was 22.8+/-11.2 h (xh +/- pseudo-SD), peak plasma concentration cmax approximately 30 microg/ml at a dose of 420 mg/m2, distribution volume Vss= 5.34+/-2.1 L (xa +/- SD), and a total clearance CItotal = 2.58+/-1.23 ml/min (xa +/- SD). There was a linear correlation between the area under the curve(0-infinity) of Ti in plasma and the titanocene dichloride dose administered with a correlation coefficient r2 of 0.8856. Plasma protein binding of Ti was in the 70-80% range. Between 3% and 16% of the total amount of Ti administered were renally excreted during the first 36 h. The recommended dose for Phase II evaluation is 240 mg/m2 given every 3 weeks with i.v. hydration to reduce renal toxicity.

Phase I trial of weekly scheduling and pharmacokinetics of titanocene dichloride in patients with advanced cancer.

PURPOSE: To determine the maximum-tolerated dose (MTD) and the dose-limiting toxicities (DLTs) of a weekly schedule of titanocene dichloride (TD) and to define the pharmacokinetics of titanium in plasma and urine. PATIENTS AND METHODS: Twenty patients with a median age of 58 years received 83 courses of TD. TD was given as 1-hour infusion at escalating doses from 70 to 185 mg/m2/wk. Pharmacokinetic analysis was performed in eight patients for total plasma titanium (TPTi) and in three patients for ultrafiltrable titanium (UFTi). RESULTS: At the fifth dose level (185 mg/m2/wk), a variety of DLTs were seen in five patients: fatigue in three, bilirubinemia in one, and hypokalemia in two. A further six patients were treated at 140 mg/m2; only one had dose-limiting creatinine elevation and this dose was therefore defined as the MTD. No myelosuppression or alopecia were observed. One patient with adenocarcinoma of unknown primary had a minor response. Pharmacokinetic analysis showed that TPTi maximum concentration (Cmax) values were linear with dose and elimination of TPTi was triphasic with a long terminal half-life (t1/2; median, 165 hours; range, 89 to 592). Between 7% and 24.3% of the total of administered titanium was eliminated in urine over the first 24 hours. In contrast, UFTi elimination was described by a one-compartment model with a t1/2 of 0.41 hours; peak levels of UFTi were 5.2% +/- 2.5% those of TPTi. CONCLUSION: The MTD of TD given on a weekly schedule is 140 mg/m2, with cumulative, but reversible creatinine and bilirubin elevation being the DLTs.

Comparative analysis of position-effect variegation mutations in Drosophila melanogaster delineates the targets of modifiers.

In Drosophila melanogaster, heterochromatin-induced silencing or position-effect variegation (PEV) of a reporter gene has provided insights into the properties of heterochromatin. Class I modifiers suppress PEV, and class II modifiers enhance PEV when the modifier gene is present in fewer than two doses. We have examined the effects of both class I and class II modifiers on four PEV mutations. These mutations include the inversions In(1)w(m4) and In(2R)bw(VDe2), which are classical chromosomal rearrangements that typify PEV mutations. The other mutations are a derivative of brown(Dominant), in which brown+ reporters are inactivated by a large block of heterochromatin, and a P[white+] transposon insertion associated with second chromosome heterochromatin. In general, we find that class I modifiers affect both classical and nonclassical PEV mutations, whereas class II modifiers affect only classical PEV mutations. We suggest that class II modifiers affect chromatin architecture in the vicinity of reporter genes, and only class I modifiers identify proteins that are potentially involved in heterochromatin formation or maintenance. In addition, our observations support a model in which there are different constraints on the process of heterochromatin-induced silencing in classical vs. nonclassical PEV mutations.

Antitumor activity of new organometallic compounds in human ovarian cancer cell lines and comparison to platin derivatives.

Cisplatin, Carboplatin and new compounds such as Paclitaxel and Docetaxel are effective drugs in the treatment of ovarian cancer. Multidrug resistance however remains an issue in ovarian cancer. The search for new effective drugs will remain of the highest priority in the field of cancer research. The in vitro and in vivo growth inhibiting potencies of two new metallocene dichlorides, Titanocendichloride and Vanadocendichloride, were compared to Cisplatin and Carboplatin using 20 permanent human ovarian cancer cell lines. Under in vivo and in vitro conditions Cisplatin was more effective than Carboplatin. Under in vitro conditions a Vanadocendichloride concentration of as low as 1.5 x 10(-7) mol/l resulted in a 50% decrease in the cell proliferation. Titanocendichloride inhibited cellular growth only at concentrations of 10(-4) mol/l. In contrast, Titanocendichloride was more effective in inhibiting growth of xenotransplanted ovarian cancer cell lines in nude mice. The clinically supposed equipotentiality of Carboplatin should be handled cautiously. The new organometallic substances clearly exhibit antiproliferative properties in ovarian cancer cells and are considered for clinical phase I trials.

The N-terminus of Nef from HIV-1/SIV associates with a protein complex containing Lck and a serine kinase.

The Nef protein of human and primate lentiviruses is a key factor in HIV/SIV pathogenesis. Here we report that Nef associates with two different kinases, forming a multiprotein complex at the far N-terminus of the viral protein. One of the kinases was identified as Lck, whereas the second protein was found to be a serine kinase that phosphorylated Nef and Lck in vitro and could be discriminated from the serine kinase identified previously. The Nef-associated kinase complex (NAKC) was demonstrated in COS cells, in HIV-infected cells, and in vitro using recombinant Lck and Nef proteins. Deletion of a short amphipathic alpha-helix in the N-terminus, which was found to be conserved in all Nef proteins, inhibited association of the NAKC and significantly reduced virion infectivity.

Antitumor activity of treosulfan in human lung carcinomas.

Treosulfan (L-threitol 1,4-bismethanesulfonate, Ovastat) is an alkylating agent and a structural analogue of busulfan. It has been established in the clinical chemotherapy of human ovarian carcinomas for several years and has additionally been shown to be effective against xenografted human breast carcinomas. No other human carcinoma is yet known to be sensitive to treosulfan. The present study confirms the pronounced and significant antitumor activity of treosulfan against heterotransplanted human lung carcinomas of both the small-cell and the non-small-cell type. Treosulfan reduced the growth of all four small-cell lung carcinomas that were investigated in a significant manner. It was even more active than equitoxic doses of the clinically approved cytostatics ifosfamide, cisplatin, and etoposide toward three of them and induced long-lasting growth reductions (60-98% of control tumor size) corresponding to partial and nearly complete remissions. In the case of the nine non-small-cell lung carcinomas investigated, treosulfan effected significant growth inhibition of more than 50%, again in all of them, and was more active than the comparative compounds ifosfamide, mitomycin C, and cisplatin at least in one of four epidermoid lung carcinomas, one large-cell carcinoma, and one of three lung adenocarcinomas. These results are remarkable and unexpected, and the present study should be followed rapidly by phase II clinical trials of treosulfan against human lung carcinomas of both the small-cell and the non-small-cell type.

In vitro activity of titanocenedichloride versus cisplatin in four ovarian carcinoma cell lines evaluated by a microtiter plate ATP bioluminescence assay.

Titanocenedichloride (MKT 4) is a novel anticancer drug with a broad spectrum of activity in mammalian tumors. We investigated the anticancer efficacy of MKT 4 versus cisplatin and its chemomodulation by buthionine sulfoximine (BSO) in four different human ovarian carcinoma (OvCA) cell lines derived from both primary (A2780. OTN 14) and recurrent tumors (SKOV-3 and OV-MZ-1b) using an in vitro microplate ATP bioluminescence assay (ATP-TCA). Sensitivity against cisplatin was higher in A2780 and OTN 14 compared with MKT 4, whereas the opposite was found in SKOV-3 and OV-MZ-1b cells. In A2780, SKOV-3 and OV-MZ-1b, the cytotoxicity of both agents could be effectively improved by BSO with supraadditive effects observed for MKT 4 in all three cell lines. In OTN 14, however, BSO treatment failed to increase the cytotoxicity of both cisplatin and MKT 4. These results suggest antineoplastic activity of MKT 4 in cisplatin-sensitive and mainly in cisplatin-resistant OvCA cells which can be significantly modulated by BSO-mediated glutathione depletion. Since antineoplastic activity of both cisplatin and MKT-4 observed in OTN 14 could not be reversed by BSO, other mechanisms of drug resistance different from the glutathione redox cycle are likely to be important for both metal compounds.