Clinical biopsychosocial risk factors for depression in lung cancer patients: a comprehensive analysis using data from the Lung Cancer Database Project.

BACKGROUND: Various risk factors for depression in lung cancer patients have been suggested but have been examined separately in studies with relatively small sample sizes. The present study examined the biopsychosocial risk factors of depression in lung cancer patients, focusing on psychological factors in the largest patient sample reported to date. PATIENTS AND METHODS: A total of 1334 consecutively recruited lung cancer patients were selected, and data on cancer-related variables, personal characteristics, health behaviors, physical symptoms, and psychological factors were obtained. The participants were divided into groups with or without depression using the Hospital Anxiety and Depression Scale. RESULTS: Among the recruited patients, 165 (12.4%) manifested depression. The results of a binary logistic regression analysis were significant (overall R2, 36.5%), and a greater risk for depression was strongly associated with psychological factors, such as personality characteristics (neuroticism) and coping style (low fighting spirit, helplessness/hopelessness, and anxious preoccupation). Although the contributions of cancer-related variables, personal characteristics, health behaviors, and clinical state were relatively low, cancer stage, cancer type, sex, and age correlated significantly with depression. CONCLUSION: Depression was most strongly linked with personality traits and coping style, and using screening instruments to identify these factors may be useful for preventive interventions.

Risk factors for regrowth of intracranial meningiomas after gamma knife radiosurgery: importance of the histopathological grade and MIB-1 index.

INTRODUCTION: The influence of histopathological grade and MIB-1 index of intracranial meningioma on the results of its radiosurgical management is not clear. The objective of the present retrospective study was to make an evaluation of these factors along with an analysis of other variables associated with progression-free survival after gamma knife radiosurgery (GKR). PATIENTS AND METHODS: Thirty-four intracranial meningiomas with known detailed histopathological diagnosis were analyzed. Tumors of WHO histopathological grades I, II, and III were diagnosed in 24, 3, and 7 cases, respectively. The median MIB-1 index was 1.3% (range: 0-31.9%). In 14 cases the MIB-1 index was 3.0% and more. In 26 cases the treatment was done at the time of tumor recurrence. Median volume of the neoplasm at the time of GKR was 4.1 mL (range: 0.4-43.1 mL). Median marginal dose was 12 Gy (range: 8-19 Gy). Median length of follow-up constituted 63 months (range: 19-132 months). RESULTS: Actuarial progression-free survival at 1, 3, 5, and 10 years constituted 100, 94, 83, and 58%, respectively. Histopathological grade II or III (p<0.0001), MIB-1 index 3% and more (p=0.0004), and non-skull base location (p=0.0026) of the tumor showed negative associations with progression-free survival in multivariate analyses. Actuarial progression-free survival at 5 years after GKR for benign and non-benign meningiomas constituted 100 and 45%, respectively (p<0.0001). CONCLUSION: Radiosurgery is a highly effective management option for benign intracranial meningiomas, but growth control of non-benign ones is significantly worse. It requires close neuroradiological follow-up and necessitates the search for modified treatment strategies.

Identification of homozygous deletions of tumor suppressor gene FAT in oral cancer using CGH-array.

Homozygous deletions (HD) provide an important resource for identifying the location of candidate tumor suppressor genes. To identify the tumor suppressor gene in oral cancer, we employed high-resolution comparative genomic hybridization (CGH)-array analysis. We identified a homozygous loss of FAT (4q35), a new member of the human cadherin superfamily, from genome-wide screening of copy number alterations in one primary oral cancer. This result was evaluated by genomic polymerase chain reaction in 13 oral cancer cell lines and 20 primary oral cancers and Southern blot in the cell lines. We found frequent exonic HD of FAT in the cell lines (3/13, 23%) and in primary oral cancers (16/20, 80%). FAT expression was absent in these cell lines. Homozygous deletion hot spots were observed in exon 1 (9/20, 45%) and exon 4 (7/20, 35%). Moreover, loss of gene expression was identified in other types of squamous cell carcinoma. The methylation status of the FAT CpG island in squamous cell carcinomas correlated negatively with its expression. Our results identify mutations in FAT as an important factor in the development of oral cancer and indicate the importance of FATs function in some squamous cell carcinomas.

Induction by bufalin of differentiation of human leukemia cells HL60, U937, and ML1 toward macrophage/monocyte-like cells and its potent synergistic effect on the differentiation of human leukemia cells in combination with other inducers.

We have recently demonstrated that bufalin is a new potent inducer of the differentiation of human myeloid leukemia cells. The present work was carried out to examine further the effect of bufalin on the growth and characteristics of human leukemia-derived cell lines U937, ML1, and HL60. At concentrations of 5-10 nM, bufalin decreased the growth of ML1 cells preferentially at the G2 phase and U937 cells at the S and G2 phases of the cell cycle. Bufalin, under these conditions, induced the differentiation of U937, ML1, and HL60 cells to monocyte/macrophage-like cells by measuring the expression of various differentiation markers, as assessed by morphology and histochemistry, and ability to phagocytose latex particles, to reduce nitroblue tetrazolium, and to develop Fc receptors. U937 and ML1 cells started to differentiate at 4 and 6 h, respectively, after treatment with 10 nM bufalin and showed maximum differentiation 72 h later. At present, a mechanism for the bufalin-mediated induction of the differentiation of these human leukemia cells remains to be determined. The combination of bufalin with all-trans retinoic acid, 1 alpha,25-dihydroxyvitamin D3, 4'-demethylepipodophyllotoxin ethylidene-beta-D-glucoside (VP16), or human gamma-interferon synergistically induced the differentiation of HL60 and U937 cells. A similar effect on ML1 cells was observed with the combination of bufalin with VP16 or human rTNF-alpha. These results suggest that bufalin in combination with VP16, all-trans retinoic acid, 1 alpha,25-dihydroxyvitamin D3, rTNF-alpha, or gamma-interferon may be very useful in the differentiation of human leukemia.

Purification of a low molecular weight factor that induces differentiation and inhibits growth in myeloid leukemia cells.

A procedure is described for purifying a low molecular weight peptide that induces differentiation in mouse myeloid leukemia M1 cells. The factor comes from the conditioned medium of macrophage-like cells differentiated from mouse myeloid leukemia M1 cells. The procedure for purification includes gel filtration on Sephadex G-15, anionic exchange chromatography, thin-layer chromatography, reverse-phase high-performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography gel filtration. The molecular weight of the factor estimated from the amino acid composition was approximately 1280, which agrees well with that obtained by high-performance liquid chromatography gel filtration. The half-maximal concentration of the purified factor for inducing differentiation of M1 cells was approximately 3.2 x 10(-7) M as judged by nitroblue tetrazolium staining ability. The purified factor also inhibits the growth of human leukemia HL-60 cells.

Bcl-2 protein inhibits bufalin-induced apoptosis through inhibition of mitogen-activated protein kinase activation in human leukemia U937 cells.

In a previous study, we demonstrated that bufalin, which is an active principle of Chinese medicine, chan'su, caused apoptosis in human leukemia U937 cells by anomalous activation of mitogen-activated protein kinase (MAPK) via the signaling pathway of Ras, Raf-1, and MAPK kinase-1. Here, we report the effect of overexpression of bcl-2 in U937 cells on the signaling pathway of apoptosis that is induced by bufalin. The results indicated that the apoptosis induced by bufalin in U937 cells was significantly inhibited by overexpression of the Bcl-2 protein. No significant difference was detected in the activation of MAPK kinase-1 that is induced by bufalin in wild-type or Bcl-2-overexpressed U937 cells; however, the activation of MAPK by bufalin was significantly attenuated in the cells overexpressing Bcl-2. Bufalin treatment activated activator protein-1 transcriptional activity; however, this activation was decreased to 40% in bcl-2-overexpressed U937 cells. These results indicate that Bcl-2 acts downstream of MAPK kinase-1 but upstream of MAPK and suggest that, in the signaling pathway of the apoptotic process induced by bufalin, the transcriptional activity of activator protein-1 may be down-regulated through the inhibition of MAPK activity by Bcl-2.

Characterization of protein phosphorylation of the cytosol of AH-66 hepatoma ascites cells.

Endogenous phosphorylation reaction of the cytosol fraction of AH-66 hepatoma ascites cells in vitro was compared with that of normal rat liver. Cytosolic proteins with molecular weights of 125,000, 98,000, and 40,000 of AH-66 cells were heavily phosphorylated in a cyclic adenosine 3':5'-monophosphate-independent manner, but no counterpart was detected in normal liver cytosol. In order to examine whether these phosphoproteins were specifically present in AH-66 cytosol, cyclic adenosine 3':5'-monophosphate-independent protein kinases which phosphorylate these phosphoproteins were partially purified from AH-66 and liver cytosol by successive chromatography. Both kinase preparations were essentially free of endogenous protein substrates and catalyzed the phosphorylation of exogenous substrates, such as casein and phosvitin, but not histone and protamine. Both kinases markedly catalyzed the phosphorylation of the Mr 125,000, 98,000, and 40,000 proteins in AH-66 cytosol. The Mr 125,000, 98,000 and 40,000 proteins in liver cytosol were less intensely phosphorylated by the addition of the kinase from AH-66 cytosol. From these results, we conclude that these phosphoproteins are present in both AH-66 cytosol and liver cytosol but are highly concentrated in AH-66 cytosol.

Isolation and some biochemical characteristics of nuclei from AH-66 hepatoma cells.

Cetylpyridinium chloride uniquely facilitated the isolation of nuclei from AH-66 hepatoma ascites cells in an isotonic medium without homogenization because of its strong solubilization of their plasma membranes, which were resistant to mechanical shearing with the commonly used nonionic detergents such as Triton X-100, Nonidet P-40, and Tween 80. Virtually all the nuclei in a population of AH-66 cells (10(6)/ml) can be isolated with 0.2% cetylpyridinium chloride. The isolated nuclei were free of adherent cytoplasm, maintained satisfactory morphology, and had high activity of nicotinamide adenine dinucleotide pyrophosphorylase. Two-dimensional polyacrylamide gel electrophoresis of the acid-soluble nuclear proteins of the AH-66 hepatoma nuclei isolated by the cetylpyridinium chloride procedure as well as by the citric acid procedure revealed that Spots Ac and C16-C18 were significantly intense in the gel pattern. Unexpectedly, Spot A10 was absent from the gel pattern of AH-66 hepatoma nuclei.

Activation of AP-1 is required for bufalin-induced apoptosis in human leukemia U937 cells.

In a previous study, we demonstrated that bufalin caused apoptosis in human leukemia U937 cells by the anomalous activation of mitogen-activated protein kinase (MAPK) via a signaling pathway that included Ras, Raf-1 and MAPK kinase-1. We report here the effect of bufalin on c-Jun N-terminal protein kinase (JNK), a member of the MAPK family, and on the signaling pathway downstream of MAPKs in U937 cells. When U937 cells were treated with 10(-8) M bufalin, the activity of JNK1 was markedly elevated 3 h after the start of treatment and remained so for 9 h. This activation of JNK and the induction of apoptosis by bufalin were suppressed by expression of antisense mRNA for MAPK kinase-1. c-Jun was translocated from the cytoplasm to the nucleus after treatment of U937 cells with bufalin. The transcriptional activity of AP-1 was transiently enhanced by the treatment with bufalin and this activation was suppressed by the expression of antisense mRNA for MAPK kinase-1. Both curcumin (1,7-bis[4-hydroxy-3-methoxy-phenyl]-1,6-heptadiene-3,5-dione), an inhibitor of the biosynthesis of AP-1, and the expression of dominant negative c-Jun inhibited the activation of AP-1 and the induction of apoptosis by bufalin. Expression of a constitutively active mutant form of MAPK kinase-1 induced the activation of AP-1 and subsequent apoptosis in U937 cells. These results suggest that the activation of AP-1 via a MAPK cascade that includes JNK is required for the induction of apoptosis by bufalin in U937 cells.

Tiam1 is involved in the regulation of bufalin-induced apoptosis in human leukemia cells.

Bufalin, a component of the Chinese medicine chan'su, induces apoptosis in various lines of human tumor cells, such as leukemia HL60 and U937 cells, by altering the expression of apoptosis-related genes, for example, bcl-2 and c-myc. In this study, we characterized a gene that is involved in bufalin-induced apoptosis by the differential display (DD) technique. The partial nucleotide sequence of one of the differentially expressed clones obtained after treatment with bufalin was identical to that of the human gene for Tiam1. When U937 cells were treated with 10(-7) M bufalin, expression of both Tiam1 mRNA and the protein was induced 1 h after the start of the treatment. The increase of Tiam1 mRNA was transient but the level of Tiam1 protein continued to increase at least for 6 h. In addition, the activities of Rac1 and p21-activated kinase (PAK) were also stimulated by bufalin treatment. To evaluate the role of Tiam1 in the apoptotic process, we examined the effects of the expression of sense and antisense RNA for Tiam1 in U937 cells. Apoptosis was strongly induced by bufalin in cells that expressed sense RNA for Tiam1 as compared to apoptosis in control cells treated with bufalin only. Cells expressing antisense RNA for Tiaml were significantly more resistant than the control bufalin-treated cells to induction of DNA fragmentation in response to bufalin. Moreover, sense transformants had elevated activities of PAK and c-Jun NH2-terminal kinase (JNK). These results suggest that Tiaml might play a critical role in bufalin-induced apoptosis through the activation of Rac1, PAK, and JNK pathway.

CRBP suppresses breast cancer cell survival and anchorage-independent growth.

We showed earlier that cellular retinol-binding protein (CRBP) expression is downregulated in a subset of human breast cancers. We have now investigated the outcome of ectopic CRBP expression in MTSV1-7 cells, a SV40 T antigen-transformed human breast epithelial cell line devoid of endogenous CRBP expression. We found that: (i) CRBP did not inhibit adherent cell growth but suppressed foci formation in post-confluent cultures and colony formation in soft agar; (ii) this effect was due to CRBP inhibition of cell survival, as demonstrated by viability and TUNEL assays of cells in soft-agar or plated on polyHEMA-coated dishes; (iii) CRBP inhibited protein kinase B/Akt activation in cells in suspension but not in adherent cells and the CRBP suppression of anchorage-independent growth was mimicked by cell treatment with the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002; (iv) CRBP enhanced retinyl ester formation and storage but did not regulate retinoic acid synthesis or retinoic acid receptor activity. Ectopic CRBP-mediated inhibition of anchorage-independent cell survival and colony formation in the absence of significantly altered responses to either retinol or retinoic acid was also documented in T47D human breast cancer cells. In conclusion, the data suggest two novel and linked CRBP functions in mammary epithelial cells: inhibition of the PI3K/Akt survival pathway and suppression of anchorage-independent growth.

Isolation and partial characterization of the major glycoprotein from the plasma membranes of AH-66 hepatoma cells.

A major glycoprotein of the plasma membranes of AH-66 hepatoma ascites cells was isolated in essentially pure form and in milligram amounts. The plasma membranes were solubilized with a solution containing both 0.3 M lithium diiodosalycylate and 0.2% cetylpyridinium chloride, and further extracted with 50% phenol, followed by gel filtration on Sepharose 6B in the presence of 0.1% Ammonyx-LO at pH 8.0. The apparent molecular weight of the purified glycoprotein was estimated to be 165 000 in 5.6% polyacrylamide gels, of which 54% was carbohydrate and 46% was protein. The chemical composition of the glycoprotein resembles glycophorin A from human erythrocyte membranes in that it has a high content of N-acetylgalactosamine, N-acetylglucosamine, galactose and sialic acid and a particularly large proportion of serine, threonine, aspartic acid and glutamic acid.

Interaction between proteins and detergents which contain a hydrocarbon chain longer than 16 carbon atoms. II. Difference spectra of various proteins in cetyldimethyl-benzylammonium chloride.

The detergents which contain a hydrocarbon side chain longer than 16 cabron atoms were used as a perturbant for the study of protein structure. ta low concentration of cetyldimethylbenzylammonium chloride (CDBA) caused difference spectra for Ac-Trp-OEt and AC-Tyr-OEt. The delta e values at their difference maxima became constant above 30 mM of cetyldimethylbenzylammonium chloride, 1430 at 294 nm for Ac-Trp-OEt and 450 at 288 nm for Ac-Tyr-OEt. These delta e values are higher than any other delta e values resulting from solvent effects by such a remarkably low concentration of organic reagents described in the literature so far. The absence of denaturation blue shift in the difference spectra and the fact that the optical rotatory dispersion of the proteins examined in the present study was not changed significantly by cetyldimethylbenzylammonium chloride indicate that the secondary and tertiary structures of the proteins were not destroyed by cetyldimethylbenzylammonium chloride. These characteristics, together with small overlapping of their difference spectra at 288 and 294 nm were advantageous in the determination of tryptophan and tyrosine residues exposed in glucagon, insulin and alcohol dehydrogenase from yeast. No tyrosine residues in ribonuclease A was accessible to cetyldimethylbenzylammonium chloride. Unusual difference spectrum with a peak at 298 nm was observed for lysozyme which is known to contain tryptophan residues in special environments. Ovalbumin gave a novel unusual difference spectrum with a peak at 290 nm and a shoulder at 298 nm, showing the existence of unusual tryptophan and probably tyrosine residues in the molecule.

Interaction between proteins and detergents which contain a hydrocarbon chain longer than 16 carbon atoms. III. Competitive inhibition of trypsin by cetyldimethylbenzylammonium chloride.

Tryptic activity was competitively inhibited by cationic detergents which contain a cetyl group or longer hydrocarbon chains. Since the cetyl group is much longer than the side chains of lysine or arginine residues of substrates for trypsin, the nature of inhibition by cetyldimethylbenzylammonium chloride was examined using Na-benzoyl-L-arginine-p-nitroanilide as substrate and compared to that by butylamine. The inhibition by cetyldimethylbenzylammonium chloride occurred instantaneously and was completely reversible. The inhibitory effect of cetyldimethylbenzylammonium chloride was strongly dependent on both pH and salt concentration, contrasting with inhibition by butylamine which was relatively indifferent to these changes. The Ki value of cetyldimethylbenzylammonium chloride at pH 7.5 and 25 degrees C was calculated to be 2.0 +/- 0.3 mM, which is equal to that of butylamine within experimental errors. The standard entropy change of binding of cetyldimethylbenzylammonium chloride (44 +/- 2 cal/mol/degree) was much larger than for butylamine, indicating the formation of an efficient hydrophobic bond between the cetyl group and the enzyme.

Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells.

We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.

Fluorescence changes of rhodamine 6G associated with changes in membrane potential in synaptosomes.

The intensity of rhodamine 6G fluorescence was found to be a useful scale for measuring the membrane potential in synaptosomes. The fluorescence of rhodamine 6G in synaptosomal suspensions increases with depolarization in the synaptosomes induced by the replacement of cations in the medium or by the addition of agents known to depolarize the membrane potential. Considering the character of the dye, we have derived an equation which gives the relation between the fluorescence intensity of the dye and the membrane potential. The change in membrane potential (diffusion potential) of synaptosomes was calculated using the equation. The calculated membrane potential was proportional to the logarithm of the K+ concentration above 20 mM, and the slope of membrane potential against log [K+] was about 52 mV per decade of concentration. The permeability ratio (Px/Pk; the ratio of the permeability constants of a given cation, X+, and K+) was estimated from the calculated membrane potential.

Effects of probes of membrane potential on metabolism in synaptosomes.

Effects of three probes for measuring membrane potential, tetraphenylphosphonium (TPP+), rhodamine 6G and 3,3'-dipropylthiocarbocyanine (diS-C3-(5)) on energy metabolism in synaptosomes were investigated. None of the three probes had any effect on lactate production in synaptosomes. TPP+ and rhodamine 6G did not inhibit the respiration of synaptosomes with pyruvate and succinate as exogenous substrate and were only weakly inhibitory with endogenous substrates. In contrast, diS-C3-(5) markedly inhibited the respiration of synaptosomes with glucose, pyruvate and endogenous substrates. All three probes reduced ATP content in synaptosomes and depolarized the membrane potential in synaptosomes with increasing concentrations of the probes. It is, therefore, preferable to estimate membrane potential with TPP+ or rhodamine 6G at their low concentrations where their effect on metabolism is negligible.

Determination of disulfide array and subunit structure of taste-modifying protein, miraculin.

The taste-modifying protein, miraculin (Theerasilp, S. et al. (1989) J. Biol. Chem. 264, 6655-6659) has seven cysteine residues in a molecule composed of 191 amino acid residues. The formation of three intrachain disulfide bridges at Cys-47-Cys-92, Cys-148-Cys-159 and Cys-152-Cys-155 and one interchain disulfide bridge at Cys-138 was determined by amino acid sequencing and composition analysis of cystine-containing peptides isolated by HPLC. The presence of an interchain disulfide bridge was also supported by the fact that the cystine peptide containing Cys-138 showed a negative color test for the free sulfhydryl group and a positive test after reduction with dithiothreitol. The molecular mass of non-reduced miraculin (43 kDa) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was nearly twice the calculated molecular mass based on the amino acid sequence and the carbohydrate content of reduced miraculin (25 kDa). The molecular mass of native miraculin determined by low-angle laser light scattering was 90 kDa. Application of a crude extract of miraculin to a Sephadex G-75 column indicated that the taste-modifying activity appears at 52 kDa. It was concluded that native miraculin in pure form is a tetramer of the 25 kDa-peptide and native miraculin in crude state or denatured, non-reduced miraculin in pure form is a dimer of the peptide. Both tetramer miraculin and native dimer miraculin in crude state had the taste-modifying activity.

Enhancement of the uptake of 1-methyl-4-phenylpyridinium ion (MPP+) in mitochondria by tetraphenylboron.

The uptake of 1-methyl-4-phenylpyridinium (MPP+) by intact mitochondria was measured by an electrode sensitive to MPP+. The electrode was constructed with a polyvinyl chloride membrane that contained tetraphenylboron (TPB) as an ion-exchange. MPP+ was taken up by mitochondria in an energy-dependent process. TPB rapidly enhanced MPP+ uptake by mitochondria, and then induced release of MPP+ from mitochondria in medium containing glutamate and malate. No release of MPP+ from mitochondria after addition of TPB could be observed in medium containing succinate, the oxidation of which is not inhibited by MPP+. The release of MPP+ was caused by respiratory inhibition by MPP+ taken up in mitochondria. Since the release of MPP+ did not increased O2 uptake in mitochondria, the major part of MPP+ released from the matrix, where no respiratory enzyme inhibited by MPP+ exists. We concluded the following effect of TPB on MPP+ uptake from the results: (1) The increase of MPP+ concentration in matrix by addition of TPB increased the amount of bound to the inner membranes of mitochondria. (2) The increase of the amount of MPP+ in the inner membranes enhanced the respiratory inhibition. (3) The respiratory inhibition induced to release MPP+ from the matrix. The relation between MPP+ distribution in the membrane of mitochondria and the respiratory inhibition by MPP+ are discussed.

An enzyme immunoassay and immunoblot analysis for curculin, a new type of taste-modifying protein: cross-reactivity of curculin and miraculin to both antibodies.

We have developed an enzyme immunoassay method for curculin, a new type of taste-modifying protein. This method can accurately quantify 0.05-20 ng of curculin, a sensitivity about 3000-times that of the psychometric method. The content of curculin in the fruit of Curculigo latifolia increased gradually until 3 weeks after artificial pollination and dramatically at 4 weeks, to finally reach 1.3 mg per fruit. Immunoblot analysis indicated that antiserum to curculin was faintly reactive with miraculin, but not with thaumatin or monellin.