Breast tumor kinase (Brk/PTK6) plays a role in the differentiation of primary keratinocytes.

Breast Tumor Kinase (Brk/PTK6) has a relatively limited expression profile in normal tissue. Its expression is restricted to epithelial cells that are differentiating such as those in the epidermis, and Brk expression appears to be absent from proliferating cells in normal tissue. Also, there is now some evidence to suggest that Brk plays a functional role in the differentiation of the keratinocytes in the epidermis. We have, therefore, investigated the role that Brk/PTK6 plays in normal human primary keratinocytes by suppressing protein levels using RNA interference. We show that as primary human keratinocytes are induced to differentiate in vitro, Brk levels decrease. Decreasing Brk protein levels lead to an increase in the number of cells with a permeable plasma membrane, a decrease in epidermal growth factor receptor (EGFR) and a parallel increase in keratin 10 levels, but classical markers of apoptosis or terminal differentiation are not affected. We propose Brk, Keratin 10 and EGFR are co-regulated during differentiation and that manipulating Brk expression can influence the differentiation of normal primary human keratinocytes.

In vivo characterization of horseradish peroxidase with indole-3-acetic acid and 5-bromoindole-3-acetic acid for gene therapy of cancer.

Gene-directed enzyme prodrug therapy is a form of targeted cancer therapy, in which an enzyme is used to convert a non-toxic prodrug to a cytotoxin within the tumor. Horseradish peroxidase (HRP) is able to convert the indole prodrugs indole-3-acetic acid (IAA) and the halogenated derivative 5-bromo-IAA (5Br-IAA) to toxic agents able to induce cell kill in vitro. This study characterized HRP-directed gene therapy in vivo. Human nasopharyngeal squamous cell carcinoma cells, FaDu, stably expressing HRP were grown as xenografts in SCID mice. Pharmacokinetic analysis of IAA and 5Br-IAA showed satisfactory drug profiles, and millimolar concentrations could be achieved in tumor tissue at non-toxic doses. HRP-expressing tumors showed a modest growth delay when treated with IAA compared with drug-vehicle controls. Treatment response could not be improved using different drug scheduling or drug vehicle, nor by combining HRP-directed gene therapy with fractionated radiotherapy.

Use of horseradish peroxidase for gene-directed enzyme prodrug therapy with paracetamol.

Gene therapy is a potential method of treating cancer with a greater degree of targeting than conventional therapies. In addition, therapy can be directed towards cells within the tumour population that are traditionally resistant to current treatment schedules. Horseradish peroxidase (HRP) can oxidise paracetamol to N-acetyl-p-benzoquinoneimine via a one-electron pathway. Incubation of human cells expressing HRP with 0.5-10 mM paracetamol reduced clonogenic survival, but had little effect on control cells. A small increase in apoptosis was seen and a decrease in the number of cells undergoing mitosis, consistent with reports in hepatocytes using higher paracetamol concentrations. The cytotoxicity was also seen under conditions of severe hypoxia (catalyst induced anoxia), indicating that the HRP/paracetamol combination may be suitable for hypoxia-targeted gene therapy.

A constitutive cytoprotective pathway protects endothelial cells from lipopolysaccharide-induced apoptosis.

Lipopolysaccharide (LPS) has been implicated as the bacterial component responsible for much of the endothelial cell injury/dysfunction associated with Gram-negative bacterial infections. Protein synthesis inhibition is required to sensitize the endothelium to lipopolysaccharide-induced apoptosis, suggesting that a constitutive or inducible cytoprotective protein(s) is required for endothelial survival. We have identified two known endothelial anti-apoptotic proteins, c-FLIP and Mcl-1, the expression of which is decreased markedly in the presence of cycloheximide. Decreased expression of both proteins preceded apoptosis evoked by lipopolysaccharide + cycloheximide. Caspase inhibition protected against apoptosis, but not the decreased expression of c-FLIP and Mcl-1, suggesting that they exert protection upstream of caspase activation. Inhibition of the degradation of these two proteins with the proteasome inhibitor, lactacystin, prevented lipopolysaccharide + cycloheximide-induced apoptosis. Similarly, lactacystin protected against endothelial apoptosis induced by either tumor necrosis factor-alpha or interleukin-1beta in the presence of cycloheximide. That apoptosis could be blocked in the absence of new protein synthesis by inhibition of the proteasome degradative pathway implicates the requisite involvement of a constitutively expressed protein(s) in the endothelial cytoprotective pathway. Finally, reduction of FLIP expression with antisense oligonucleotides sensitized endothelial cells to LPS killing, demonstrating a definitive role for FLIP in the protection of endothelial cells from LPS-induced apoptosis.

Nociceptin and the ORL-1 ligand [Phe1psi (CH2-NH)Gly2]nociceptin(1-13)NH2 exert anti-opioid effects in the Freund's adjuvant-induced arthritic rat model of chronic pain.

1 Stimulation of the opioid receptor-like1 (ORL-1) receptor by nociceptin (NC) produces hyperalgesia and reverses the antinociceptive effects induced by opioids. Most studies concerning the central effects of NC were conducted using acute pain models. The role NC may play in chronic inflammation remains unelucidated. 2 The present study was undertaken to assess the action of NC in the Freund's adjuvant-induced monoarthritic rat model. The effects of drugs known to act as analgesics in this model were evaluated. The effects of NC, NCNH2, and the ORL-1 ligand, [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 ([F/G]NC(1-13)NH2), were also studied alone or in association with morphine. 3 NC (1 - 30 nmol, i. c.v.) was inactive, whilst NCNH2 (10 nmol, i.c.v.) exerted hyperalgesic effects (-4.5+/-0.9 vs -0.7+/-0.8 s of vehicle-treated animals). [F/G]NC(1-13)NH2 (0.01 - 10 nmol, i.c.v.) induced hyperalgesia in the arthritic paw (-3.3+/-0.6 vs -0.3+/-0.5 s of vehicle-treated animals; 10 nmol). 4 Both NC (0.01 - 10 nmol, i.c.v. ) and [F/G]NC(1-13)NH2 (0.01 - 1 nmol, i.c.v), 30 min after morphine (3 mg kg-1, s.c.) induced an immediate and short-lived reversal of morphine effects (2.6+/-0.3 vs 10.4+/-1.0 and 1.2+/-1.5 vs 9.3+/-1.1 s of morphine alone, respectively), therefore displaying anti-opioid activity. 5 In the Freund's adjuvant-induced rat model of arthritis, both NC and [F/G]NC(1-13)NH2 act as anti-opioid peptides. Furthermore, NCNH2 and [F/G]NC(1-13)NH2 induce hyperalgesia when given alone. Further investigations and the identification of a centrally acting ORL-1 antagonist are necessary to better understand the role of NC in pain mechanisms.

NF-kappaB activation is required for human endothelial survival during exposure to tumor necrosis factor-alpha but not to interleukin-1beta or lipopolysaccharide.

In the presence of a protein synthesis inhibitor, cycloheximide, tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1beta), or lipopolysaccharide (LPS) induces human umbilical vein endothelial cells (HUVECs) to undergo apoptosis, suggesting that constitutive or inducible cytoprotective pathways are required for cell survival. We studied the correlation between nuclear factor-kappaB (NF-kappaB) activation and cell death induced by TNF-alpha, IL-1beta, or LPS. Adenovirus-mediated overexpression of a dominant-negative IkappaBalpha (inhibitor of kappaB) mutant blocked NF-kappaB activation by gel shift assay and blocked induction of vascular cell adhesion molecule-1 protein by TNF-alpha, IL-1beta, and LPS, a NF-kappaB-dependent response. In cells overexpressing the IkappaBalpha mutant, TNF-alpha induced cell death, whereas IL-1beta or LPS did not. We conclude that cell survival following TNF-alpha stimulation is NF-kappaB-dependent but that a constitutive or inducible NF-kappaB-independent pathway(s) protects IL-1beta- or LPS-treated HUVECs from cell death.

Shear stress-mediated extracellular signal-regulated kinase activation is regulated by sodium in endothelial cells. Potential role for a voltage-dependent sodium channel.

Fluid shear stress is an important regulator of endothelial cell (EC) function. To determine whether mechanosensitive ion channels participate in the EC response to shear stress, we characterized the role of ion transport in shear stress-mediated extracellular signal-regulated kinase (ERK1/2) stimulation. Replacement of all extracellular Na+ with either N-methyl-D-glucamine or choline chloride increased the ERK1/2 stimulation in response to shear stress by 1.89 +/- 0.1-fold. The Na+ effect was concentration-dependent (maximal effect,

Effect of immunosuppressive therapy, serum creatinine, and time after transplant on plasma total homocysteine in patients following heart transplantation.

OBJECTIVES: To determine the prevalence of hyperhomocysteinemia in heart transplant recipients, and to assess the effect of renal function and immunosuppressive medication on total plasma homocysteine (tHcy) levels. BACKGROUND: Elevated plasma tHcy levels have been associated with increased risk of mortality in patients with established coronary artery disease. Graft coronary disease is the major cause of morbidity and mortality in long-term survivors of heart transplantation. The tHcy has been found to be elevated in heart and kidney transplant patients, however, the etiologic factors have not been clearly delineated. METHODS: The study group consisted of 70 heart transplant recipients (56 males, 14 females, mean age 53+/-13 years [range 17 to 69 years]). The parameters evaluated were fasting tHcy level, cumulative cyclosporine (CyA) dose, cumulative prednisone dose, serum creatinine, and time from transplantation. RESULTS: The mean fasting tHcy level was 20.5+/-10.2 micromol/L (range 5.2 to 59.0 micromol/L). Sixty-one (87%) had fasting tHcy levels greater than the seventy-fifth percentile of the general population (>12.2 micromol/L in males, and >10.1 micromol/L in females). There was no difference in mean post-transplant tHcy level between patients with and without coronary artery disease before transplantation (21.0+/-11.4 vs. 19.3+/-6.7 micromol/L, p = NS). There were significant relationships between the tHcy level and the serum creatinine (r = 0.76, p<0.001), and cumulative exposure to CyA (r = 0.31, p<0.01). There were no significant relationships between tHcy levels and cumulative prednisone dose, or time from transplantation. CONCLUSIONS: Fasting tHcy levels are markedly elevated in the majority of patients following heart transplantation, and are correlated to serum creatinine. Further studies are needed to determine other etiologic factors of elevated tHcy following heart transplantation, and to examine the impact of elevated tHcy on clinical outcomes.

A protein-binding site with dyad symmetry in the long terminal repeat of the MCF13 murine leukemia virus that contributes to transcriptional activity in T lymphocytes.

We have previously identified regions in the long terminal repeat (LTR) of the MCF13 murine leukemia virus (MLV) that contribute to transcriptional activity in different cell types. We have observed that enhancer sequences and a region that resides 3' of the enhancer make significant contributions to transcriptional activity in T lymphocytes (T. Hollon and F. K. Yoshimura, J. Virol. 63:3353-3361, 1989). In this report, we have focused on the region of the MCF13 LTR that is 3' of the enhancer to identify binding sites for proteins that may play a role in the regulation of transcription in T cells. By gel shift and DNA footprint analyses, we have identified a single protein-binding site (MLPal) that includes a nucleotide sequence with dyad symmetry. A synthetic double-stranded oligonucleotide corresponding to this protein-binding site formed a specific protein-DNA complex. Deletion of this protein-binding site from the wild-type LTR decreased transcriptional activity in T lymphocytes but not in fibroblasts as determined by a transient expression assay. The MLPal sequence by itself cannot augment transcription in T cells but is able to do so in conjunction with enhancer sequences.

Contributions to transcriptional activity and to viral leukemogenicity made by sequences within and downstream of the MCF13 murine leukemia virus enhancer.

We have identified nucleotide sequences that regulate transcription in both a cell-type-specific and general manner in the long terminal repeat of the MCF13 murine leukemia virus. Besides the enhancer element, we have observed that the region between the enhancer and promoter (DEN) has a profound effect on transcription in different cell types. This effect, however, was dependent on the copy number of enhancer repeats and was detectable in the presence of a single repeat. When two enhancer repeats were present, the effect of DEN on transcription was abrogated except in T cells. DEN also makes a significant contribution to the leukemogenic property of the MCF13 retrovirus. Its deletion from the MCF13 virus dramatically reduced the incidence of thymic lymphoma and increased the latency of disease in comparison with the wild-type virus. This effect was most marked when one rather than two enhancer repeats was present in the mutant viruses. We also observed that the removal of one repeat alone remarkably reduced leukemogenicity by the MCF13 virus. A newly identified protein-binding site (MLPal) located within DEN affects transcription only in T cells, and its deletion attenuates the ability of an MCF13 virus with a single enhancer repeat to induce thymic lymphoma. This observation suggests that the MLPal protein-binding site contributes to the effect of the DEN region on T-cell-specific transcription and viral leukemogenicity. This study identifies the importance of nonenhancer sequences in the long terminal repeat for the oncogenesis of the MCF13 retrovirus.

Differential DNA binding of nuclear proteins to a long terminal repeat region of the MCF13 and Akv murine leukemia viruses.

Long terminal repeat (LTR) sequences of murine leukemia viruses (MLVs) have been demonstrated to be mainly responsible for the pathogenic differences in these retroviruses. A region of the LTR which is downstream of the enhancer elements has been shown to contribute both to enhancer activity as well as to disease specificity of MLVs. We have identified protein-DNA complexes generated by this region of a lymphomagenic MLV (MCF13) and one which is nonpathogenic (Akv). One protein-DNA complex we have observed for this region is unique to MCF13 DNA sequences. Detection of protein involved in this unique MCF13 complex in different cell lines revealed that it was ubiquitous.

Fascicular nerve repairs. A comparative study of epineurial and fascicular (perineurial) techniques.

At present, fascicular nerve repairs produce almost identical results to epineurial repairs. There are still theoretical advantages to the improved alignment potentially offered by fascicular nerve repair, and fascicular technique already offers an advantage in partial nerve injuries. As fascicular capabilities improve, fascicular nerve repair may still offer the best solution for nerve recovery.

Diminished pentose cycle flux in perfused livers of ethanol-fed rats.

Rates of NADPH generation by the pentose phosphate pathway were evaluated in perfused livers from ethanol-fed or control rats by measuring the production of 14CO2 from 1-14C-glucose. Under basal perfusion conditions, livers from ethanol-fed rats released lactate and pyruvate into the perfusate at rates that were only 19% of the control values. Under these conditions, calculated rates of NADPH generation by the pentose cycle in livers of the ethanol-fed rats were only 50% of rates obtained with livers of control rats. 7-Ethoxycoumarin (7-EC), a substrate for mixed function oxidation, was infused to increase rates of hepatic NADPH utilization. In livers from control rats, 7-EC was oxidized at a rate of 2.6 mumol/g/hr, but rates of NADPH generation by the pentose cycle were increased by 8.8 mumol/g/hr. In livers from ethanol-fed rats, 7-EC was metabolized at rates of 7.2 mumol/g/hr, but the generation of NADPH by the pentose cycle was increased by only 3.9 mumol/g/hr. The infusion of 7-EC was associated with increases in rates of O2 uptake that exceeded rates of mixed function oxidation in both groups of animals. Ethanol feeding decreased the activity of glucose-6-phosphate dehydrogenase by 40% and decreased the concentrations of glycogen by 66%. Thus, the decrease in pentose cycle flux in perfused livers may be due to diminished activity of the rate-controlling enzyme and/or diminished substrate supply from glycogen. However, cytosolic NADP+/NADPH ratios were identical in livers of both groups. Because NADPH was not depleted during the mixed function oxidation of 7-EC in livers from ethanol-fed rats, it is concluded that other hepatic sources of NADPH compensate for the diminished generation by the pentose cycle.

7-ethoxycoumarin O-deethylation in perfused livers from ethanol-fed rats: evidence for an important role of mitochondrial reducing equivalents.

Rates of 7-ethoxycoumarin (7EC) O-deethylation in perfused livers were increased approximately 3-fold by chronic ethanol feeding. The acute addition of ethanol (5 mM) and antimycin A (0.03 mM) strongly inhibited 7EC metabolism in perfused livers from ethanol-fed rats, but less inhibition was observed when these agents were added to microsomes or to perfused livers from control rats. The activity of the hepatic pentose phosphate cycle in perfused livers was assessed by comparing 14CO2 release during the infusion of 1-14C-glucose or 6-14C-glucose. 7EC infusion caused a 3-fold greater increase in 14CO2 production from 1-14C-glucose in a liver from a control rat than in a liver from an ethanol-fed rat, indicating greater hepatic pentose cycle activity in livers of control rats. Thus, the pronounced inhibition of 7EC metabolism caused by infusion of ethanol and antimycin A may be explained by a greater dependency on mitochondrial sources of NADPH in livers of ethanol-fed rats. Dinitrophenol (0.05 mM) did not inhibit 7EC metabolism in perfused livers, indicating that a reduction in the cellular redox state, and not diminished energetics, is involved in the mechanism of inhibition produced by antimycin A.

Endothelial cell injury in vitro is associated with increased secretion of an Mr 43,000 glycoprotein ligand.

A novel, serum albumin-binding glycoprotein of molecular weight (mw) 43,000 (43K protein) was initially purified from the culture medium of bovine aortic endothelial (BAE) cells (Sage, H., Johnson, C., and Bornstein, P., J. Biol. Chem. 259:3993-4007, 1984). Its secretion by normal mesenchymal cells and by transformed cells of both ectodermal and endodermal origin suggested a general role in cellular function. To examine the effect of sublethal injury in vitro on the biosynthesis of 43K protein, BAE cells were exposed to endotoxin. At concentrations which produced minimal cell detachment and lysis, the cells secreted 70-100% more protein compared to control cultures, and the relative increase in 43K protein over total protein was approximately three-fold. A second type of cellular injury, manifested by rapid cellular proliferation and migration in response to sparse plating density (a condition that we have termed 'culture shock'), was also accompanied by a significant increase in the secretion of 43K protein. Pulse-chase studies revealed that the initial product secreted within 1.5 h was of Mr 38,000, and that between 6 and 21 h this molecule was converted to the final form of Mr 43,000. The 43K protein was not associated with RNA or glycosaminoglycan, but appeared to be linked to complex oligosaccharides containing peripheral sialosyl residues. Treatment with tunicamycin produced lower mw forms that displayed reduced affinity for albumin. By immunologic criteria, peptide mapping, and amino acid analysis, the 43K protein was shown to be structurally distinct from several proteins of Mr 40,000-50,000 associated with endothelium or with serum, including tissue factor, a plasminogen anti-activator, and several apolipoproteins. In addition, the 43K protein was not present in the extracellular matrices of endothelial, fibroblastic, or smooth muscle cells, nor was it found in plasma, serum, platelet releasate, or alveolar lavage fluids. These studies identify a unique Mr 43,000 glycoprotein that is associated with cellular stress or injury in vitro. As a secreted but nonmatrix macromolecule, this protein may be part of a 'survival kit' used by the endothelium to cope with cellular injury.

Growth factor regulation of membrane transport in human fibroblasts and its relationship to stimulation of DNA synthesis.

Serum stimulation of serum-deprived or density-inhibited normal cells enhances the level of various nutrient and ionic transport systems. Certain of these systems have been implicated in the regulation of cell proliferation. However, the use of serum stimulation to activate quiescent cells leads to enhancement of numerous transport systems with little understanding of which component or components of serum are related to activation of which transport systems. In this study we attempt to identify the specific effect of three known growth promoting factors (insulin, dexamethasone and epidermal growth factor [EGF]) on the activation of four membrane transport systems (A-amino acids, L-amino acids, glucose and K+) in normal and SV40-transformed WI38 human fibroblasts. We have also evaluated the effect of these growth factors on the stimulation of DNA synthesis in growth factor deprived cells. Thus, we can correlate the effect on a given transport system with the relative mitogenic stimulation produced by the growth factor. We conclude a) that a growth factor can effect a transport system differently in a normal versus transformed cell, b) that a specific growth factor can effect multiple transport systems and, c) with the exception of K+ transport, enhanced transport induced by a given growth factor does not necessarily correlate with the mitogenic potency of the growth factor. This latter point is of particular significance since the activation of K+ transport reflects, based on other studies, activation of the Na+-H+ exchanger which has been implicated in cell-cycle activation.