Cytotoxic action of 2-deoxy-D-glucose tetraacetate upon human lymphocytes, fibroblasts and melanoma cells

Obesity can play a significant role in chronic diseases, sudden unexpected death, and morbid obesity may be important as a cause of death for forensic pathologists. Our study attempted to determine if there is a correlation between panniculus measurements and body mass index (BMI) since BMI has been used in most studies to categorize obesity. Using data obtained from a review of 524 adult autopsies conducted at the University of Michigan from 1990 to 1992 we were able to show a correlation between both thoracic and abdominal panniculus and BMI (r2 = 0.335 and 0.296 respectively) which is statistically significant (p = 10(-47) and 10(-41) respectively). A prospective study confirmed the correlation (r2 = 0.552 for thoracic and 0.436 for abdominal panniculus) when the measurements were taken at the xyphoid process and 3 cm below the umbilicus. Using these data we calculated a panniculus index (PI) which is equal to the thoracic + abdominal panniculus in centimeters divided by the square of the height (in meters). The PI strongly correlated with BMI and was able to predict obesity. Using a BMI cutoff of 39 for morbid obesity, a PI value of 4.07 for females and 3.25 for males predicted morbid obesity with the probability of a false positive less than or equal to 2.5%. Mild and severe obesity could also be determined using the PI. Based on these data we've concluded that a concise mathematical relationship does exist between BMI and panniculus measurements. Therefore panniculus measurements can be used either as a surrogate measurement of morbid obesity or to support BMI calculations.     

Cytotoxic mechanism and antineoplastic action of etoposide

Concerned with our capacity to bridge the gap between patients' and families' psychosocial needs and the services provided, we developed a psychosocial intake and referral service. This paper will describe the lessons learned in trying to introduce an innovation whose time had not yet come and, after analyzing the outcome, to present a new approach to planning. The service was not approved and, on reflection, eight factors were identified as contributing to the failure of the service to reach fruition. In light of what we learned, we developed a new planning model that reflects planning as a circular, iterative process rather than as a linear process.     

Cytotoxic effects of MPTP on SH-SY5Y human neuroblastoma cells

Morphological and metabolic endpoints were used to evaluate MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) toxicity to SH-SY5Y human neuroblastoma cells. After 8 hours of exposure, MPTP was found to affect cell viability only at a very high concentration (3 x 10(-3) M), but its metabolite MPP+ could decrease viability at 10(-4) M. MPTP, via its metabolite MPP+, inhibited NADH dehydrogenase activity when concentrations exceeded 10(-4) M (for MPP+ 10(-5)M). The Ki were 2.4 x 10(-3) M and 3 x 10(-4)M for MPTP and MPP+, respectively. MPTP at concentrations greater than 10(-4) M altered cell morphology as early as one hour after exposure. These changes included formation of cell surface blebs and attenuated neurites. After 8 hours at 10(-3) M and 24 hrs at 10(-4) M, MPTP caused ultrastructural changes of mitochondria with increased electron-density of the matrix and disorganization of cristae, as well as abnormal aggregation of filamentous material of the cytoskeleton. Because these changes of structure and function took place at concentrations lower than those needed to affect cell viability, they may play a role in MPTP neurotoxicity in SH-SY5Y cell culture.     

Biological activity and phosphorylation of 2'-azido-2'-deoxyuridine and 2'-azido-2'-deoxycytidine

2'-Azido-2'-deoxyuridine and 2'-azido-2'-deoxycytidine were evaluated for their inhibitory activity against ribonucleotide reductase and for subsequent cell growth inhibition. Their mono- and di-phosphates were synthesized and their inhibitory activities against the reductase were also determined in a permeabilized cell system, along with the two nucleosides. The results of the present study identify the first phosphorylation step involved in the conversion of the two azidonucleosides to the corresponding diphosphates to be rate-limiting in the overall activation.     

Cytotoxic action of cis-unsaturated fatty acids on human cervical carcinoma (HeLa) cells in vitro

The effect of n-3 and n-6 fatty acids (FAs) on the growth of human cervical carcinoma (HeLa) cells was studied. Of all the FAs tested, docosahexaenoic acid (DHA, 22:6 n-3) and eicosapentaenoic acid (EPA, 20:5 n-3) were found to be the most potent in their cytotoxic action on HeLa cells and the potency of various fatty acids with regard to their cytotoxic action was as follows: DHA > EPA > dihomo-gamma-linolenic acid (DGLA) = gamma-linolenic acid (GLA) > linoleic acid (LA) > arachidonic acid (AA) > alpha-linolenic acid (ALA). The cycloxygenase inhibitor indomethacin, the lipoxygenase inhibitor nordihydroguaretic acid (NDGA), the antioxidants vitamin E, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT), the superoxide anion quencher superoxide dismutase (SOD), the hydroxyl and hydrogen peroxide quenchers mannitol and catalase, respectively, and the calmodulin antagonists trifluoperazine (TFP) and chlorpromazine (CPZ) could all block the cytotoxic action of GLA, which was used as a representative cytotoxic FA, on HeLa cells. On the other hand, copper and iron salts and buthionine sulfoxamine, a glutathione (GSH) depletor, potentiated the cytotoxic action of suboptimal doses of GLA. GLA-induced radical generation and lipid peroxidation in HeLa cells could be blocked by indomethacin, NDGA and calmodulin antagonists. The cytotoxic action of cis-unsaturated fatty acids (c-UFAs) is not dependent on the alteration in the protein kinase C levels since no alteration in the diacylglycerol levels was observed. Hydroxy and hydroperoxy products of GLA were found to be toxic to HeLa cells, whereas prostaglandin (PG)E1, PGF2 alpha, and prostacyclin stimulated cell growth. From these results, it is evident that radicals are the modulators of the cytotoxic action of c-UFAs, that their formation is a calmodulin-dependent process, and that lipoxygenase products may mediate the tumoricidal action of FAs.     

Synthesis of oligonucleotides containing two putatively mutagenic DNA lesions: 5-hydroxy-2'-deoxyuridine and 5-hydroxy-2'-deoxycytidine

Spontaneous oxidative DNA damage occurs as a consequence of aerobic metabolism, lipid peroxidation, immune responses, ionizing radiation, and some chemical oxidants. These processes yield a vast array of oxidized DNA bases and sugars. The existence of significant steady-state levels of oxidized DNA bases in the genome suggests that these lesions are not completely repaired on a biologically relevant time scale and thus may contribute to mutagenesis. In particular, studies have shown that the steady-state levels of 5-hydroxy-2'-deoxycytidine (dC5-OH) and its deamination product, 5-hydroxy-2'-deoxyuridine (dU5-OH), are similar to those found for 7,8-dihydro-8-oxoguanosine, a known highly mutagenic lesion formed by oxidation of guanosine. Structural and biological properties of dC5-OH and dU5-OH have been constrained by the lack of synthetic methodology for oligonucleotides containing these modified bases. A method is described here for the solid-phase synthesis of oligonucleotides containing dC5-OH and dU5-OH. Preparation of each of the required phosphoramidites involved the selective protection of the base 5-hydroxyl group over the deoxyribose 5'- and 3'-hydroxyl groups. The base composition and the incorporation of the adducts into synthetic heptanucleotides were confirmed after purification of the modified oligonucleotides by enzymatic digestion and HPLC analysis. Mass spectrometric analysis of the oligonucleotide products by electrospray MS and GC/MS further confirmed their composition. Most significantly, deamination of the dC5-OH oligomer to a putative dU5-OH product during solid-phase DNA synthesis or oligonucleotide deprotection was not detected by any analytical technique employed.     

Differentiation of BCS-TC2 human colon adenocarcinoma cells by sodium butyrate: increase in 5''-nucleotidase activity

Body mass index (BMI) is the most commonly used measure of obesity. Recently, some investigators have advocated direct measurement of adiposity rather than use of the BMI. This study was undertaken to determine the ability of BMI to predict body fat levels in three populations of West African heritage living in different environments. A total of 1,054 black men and women were examined in Nigeria, Jamaica, and the United States during 1994 and 1995. A standardized protocol was used to measure height, weight, waist and hip circumferences, and blood pressure at all sites; percentage of body fat was estimated using bioelectrical impedance analysis. Percentage of body fat and BMI were highly correlated within site- and sex-specific groups, and the resulting r2 ranged from 0.61 to 0.85. The relation was quadratic in all groups except Nigerian men, in whom it was linear. The regression coefficients were similar across sites, yet the mean body fat levels differed significantly (p < 0.001) as estimated by the intercept, making intersite comparison difficult. Compared with BMI, percentage of body fat was not a better predictor of blood pressure or waist or hip circumference.     

Mutagenicity of 5-aza-2'-deoxycytidine is mediated by the mammalian DNA methyltransferase

The cytosine analog 5-aza-2'-deoxycytidine has been used clinically to reactivate genes silenced by DNA methylation. In particular, patients with beta-thalassemia show fetal globin expression after administration of this hypomethylating drug. In addition, silencing of tumor suppressor gene expression by aberrant DNA methylation in tumor cells may potentially be reversed by a similar regimen. Consistent with its function in maintaining tumor suppressor gene expression, 5-aza-2'-deoxycytidine significantly reduces intestinal tumor multiplicity in the predisposed Min mouse strain. Despite its utility as an anti-cancer agent, the drug is highly mutagenic by an unknown mechanism. To gain insight into how 5-aza-2'-deoxycytidine induces mutations in vivo, we examined the mutational spectrum in an Escherichia coli lac I transgene in colonic DNA from 5-aza-2'-deoxycytidine-treated mice. Mutations induced by 5-aza-2'-deoxycytidine were predominantly at CpG dinucleotides, which implicates DNA methyltransferase in the mutagenic mechanism. C:G-->G:C transversions were the predominant class of mutations observed. We suggest a model for how the mammalian DNA methyltransferase may be involved in facilitating these mutations. The observation that 5-aza-2'-deoxycytidine-induced mutations are mediated by the enzyme suggests that novel inhibitors of DNA methyltransferase, which can inactivate the enzyme before its interaction with DNA, are needed for chemoprevention or long term therapy.     

Cloning of human Stat5B. Reconstitution of interleukin-2-induced Stat5A and Stat5B DNA binding activity in COS-7 cells

We have isolated a second human Stat5 cDNA, Stat5B, and demonstrated that the genes encoding both Stat5A and Stat5B are located at chromosome 17q11.2. Both genes were constitutively transcribed in peripheral blood lymphocytes. By using specific antisera, we demonstrated that both Stat5A and Stat5B are activated by interleukin-2 (IL-2) in peripheral blood lymphocytes, natural killer-like YT leukemia cells, and human T cell lymphotropic virus type I-transformed MT-2 T cells. In COS-7 cells, which constitutively express the Janus family tyrosine kinase Jak1, reconstitution of IL-2-induced Stat5A and Stat5B DNA binding activities was dependent on the coexpression of Jak3 along with the IL-2 receptor beta chain and the common cytokine receptor gamma-chain. This IL-2-induced Stat5 activation was dependent on the presence of either of two tyrosines (Tyr-392 or Tyr-510) in the IL-2 receptor beta chain, indicating that either of these two tyrosines can serve as a docking site. Moreover, we demonstrated that human Stat5 activation is also dependent on Tyr-694 in Stat5A and Tyr-699 in Stat5B, indicating that these tyrosines are required for dimerization. The COS-7 reconstitution system described herein provides a valuable assay for further elucidation of the IL-2-activated JAK-STAT pathway.     

Molecular modeling and mechanism of action of human decay-accelerating factor

A model of the regulatory region of human decay accelerating factor (DAF) was built based on the known coordinates of a fragment of the structurally and functionally homologous serum protein, factor H. According to this model, the four short consensus repeats (SCRs) in DAF are arranged in a helical fashion. A positively charged surface area on SCRs 2 and 3, two of the three repeating units essential for function, is postulated to be the primary recognition site for the C3 convertases C4b2a and C3bBb. This area encompasses a cavity on SCR 2, as well as part of the groove on the SCR 2-SCR 3 interface. Two additional surface depressions are centered around the C-terminal disulfide bridges of SCRs 3 and 4. These are likely to provide additional ligand binding sites. Based on this model in conjunction with sequence homology to the Ba fragment of factor B, a mechanism of DAF's accelerated convertase decay action is postulated.     

Antiproliferation activity and the mechanism of action of 9-bromo-5-morpholino-tetrazolo[1,5-c]quinazoline--potential anticancer drug

We identified 100% of the CFTR gene mutations, including three novel mutations, in 126 unrelated cystic fibrosis chromosomes from Tyrol, Austria. The frequency of the major mutation deltaF508 (74.6%) was not significantly different in Tyrolian CF-patients than in patients from Bavaria (71.0%) and Middle- and Northern Germany (71.9%), but was significantly higher than in patients from Styria (58.1%) or Northern Italy (47.6%). Interestingly, the distribution of the next most frequent mutations, R1162X (8.7%) 2183AA-->G, 2789+5G-->A and G542X (2.4% each), was more similar to the distribution of these mutations among CF-patients from Northern Italy than to those from Styria, Bavaria or Middle- and Northern Germany. Nine further mutations occurred once or twice. One of these, the missense mutation M1101K, is rare worldwide but very frequent in the Hutterite brethren, a small founder population which came from Southern Austria to Northern America. Three other different mutations (deltaL453, 1874insT and 4108delT) were present in single Tyrolian families and have not been described before. The identification of 100% of CFTR gene mutations in a particular CF population demonstrates the power of genetic analysis for the diagnosis and counselling of CF families in this restricted geographical area of Austria. Our study provides evidence for a closer genetic relation between CF patients from Tyrol and those from Bavaria or Middle- and Northern Germany as well as Northern Italy, than between CF patients from the two Austrian states Tyrol and Styria.     

Characterization of type I 5 alpha-reductase activity in DU145 human prostatic adenocarcinoma cells

Each amino acid is represented by a vector of numerical measurements for the attributes of volume, area, hydrophilicity, polarity, hydrogen bonding, shape, and charge. Inter-residue distances are then calculated according to common metrics, and we introduce a new clustering objective function derived from information-theoretic considerations. The arguments of the function are the inter-object distances of the things to be clustered: in this case the amino acids. By means of approximating the solution of an integer programming problem, then, the residues are partitioned into clusters. The clusters obtained are compared with groups obtained in substitution/mutation studies and found to be similar. Thus, probably the strongest and most objective evidence to date is supplied for believing that physico-chemical properties account for the viability of substitutions and that the important similarities/differences are explained by a relatively small and simple set of properties.     

Cytotoxic effect of Shiga toxin-1 on human proximal tubule cells

BACKGROUND: Cytolytic Shiga toxins (Stx) are believed to be largely responsible for renal damage in post-diarrheal hemolytic-uremic syndrome (D + HUS). Despite the general belief that endothelial cells are the primary target of Stx, there is evidence that proximal tubules may be a site of toxin action. We hypothesized that cultured proximal tubular cells are sensitive to the cytotoxic effects of Stx. METHODS: Cultured human proximal tubular cells were exposed to Stx-1 in the presence and absence of a variety of inflammatory factors likely to be elevated in the kidney or serum of patients with D + HUS. Cell survival, protein synthesis, total cell levels and synthesis of Stx receptors (GB3), and Stx binding were measured. RESULTS: Proximal tubules were extremely sensitive to the cytotoxic effect of Stx-1 with an LD50 at least equal to, if not less than, that seen with Vero cells. Interleukin-1 (IL-1), lipopolysaccharide (LPS), and butyrate (but not tumor necrosis factor or interleukin-6) up-regulated proximal tubule sensitivity to Stx-1. IL-1 increased Stx-1 binding, but did not alter total cell levels or synthesis of GB3, the glycosphingolipid receptor for Stx-1. In contrast, LPS and butyrate, despite increasing Stx-1 sensitivity, had no effect on Stx-1 binding. CONCLUSIONS: These studies indicate that proximal tubules are exquisitely sensitive to Stx-1 cytotoxicity and that inflammatory factors can increase toxin responsiveness through a variety of mechanisms. It is suggested that proximal tubules may be an important early target of Stx-1 action in D + HUS.     

Effect of suramin on p34cdc2 kinase in vitro and in extracts from human H69 cells: evidence for a double mechanism of action

We examined the effect of suramin, an anticancer agent and a functional analog of naturally occuring glycosaminoglycans, on p34cdc2 kinase. We find that suramin strongly inhibits the catalytic activity of purified p34cdc2 kinase (IC50 approximately 4 microM), whereas it only weakly inhibits the p13-agarose precipitated kinase activity from nuclear and cytoplasmic extracts of the asynchronous H69 human small cell lung cancer cells. We also find that the tyrosine phosphorylation of p34cdc2 kinase in the nuclear extract is increased about twice when the extracts are preincubated with 50 microM of suramin prior to the p13-agarose precipitation. We propose that this increase might result from the inhibitory effect of suramin towards p34cdc2-specific tyrosine phosphatases. These results suggest both a direct and an indirect effect of suramin on p34cdc2 kinase. We also find that heparin is a potent inhibitor of purified cdc2 kinase (IC50 approximately 3.5 micrograms/ml). Therefore, glycosaminoglycans might be physiological regulators of p34cdc2 kinase in vivo.     

Dependence of adaptative regulation for IL-1 beta action on system A activity in human synovial cells

Human synovial cells are a suitable model for estimating the physiopathological effects of IL-1 beta (IL-1) in joint. Given the importance of this cytokine in the modulation of cell metabolic activities, we set out to study the action of IL-1 on the neutral amino acid transport A system, using the methyl (aminoisobutyric) acid (MeAIB), the most highly specific and nonmetabolizable substrate for the A system. Stimulation of system A activity by adaptative regulation is a prerequisite to obtain an increase of MeAIB uptake in IL-1-treated cells, since cells which had been grown in a normal medium did not express stimulation of system A activity when IL-1 was added. The IL-1-mediated MeAIB uptake is independent of protein synthesis, since cycloheximide (CHX) did not inhibit MeAIB uptake, and characterized by a decrease in the Michaelis constant K(m) (0.147 vs. 0.270 mmol/l, IL-1 vs. control) and a slight increase in maximal velocity (Vmax) (4.59 vs. 3.89 nmol/mg prot/10 min, IL-1 vs. control). These observations indicate that IL-1 induces modifications in both system A transporter affinity and number. Moreover, we indicate that system A should be responsive in vivo to IL-1 in the same way since derepression and IL-1 action occurred in the presence of human synovial fluid.     

Phenotypic characterization of normal and CML CD34-positive cells: only the most primitive CML progenitors include Ph-neg cells

We studied the sequence of acquisition of CD33, CD38 and HLA-DR antigens on CD34+ cells from marrow and blood of Ph-chromosome positive CML patients and normal marrow. We examined the Ph status of the various CML cell populations. The mean proportions of normal and CML CD34+ cells expressing CD33 and CD38 were not significantly different. However, a significantly greater proportion of CML CD34+ cells expressed HLA-DR antigens compared with normal CD34+ cells and the level of HLA-DR expression per CML cell was abnormally high. When the sequence of acquisition of these antigens on normal and CML CD34+ cells was evaluated using 3-colour fluorescence analysis, the results suggested that HLA-DR was expressed earlier than CD38 or CD33 and these findings were confirmed by following the acquisition of CD38 and CD34+/DR+/CD38-subpopulation during liquid culture. We performed cytogenetic studies on CD34+ subpopulations in 6 cases. In 4 cases there were some Ph-negative metaphases detectable in the CD34+/DR-subpopulation (range 12.5 to 60%). In the CD34+/DR+ fractions, however, all 6 patients had only Ph-positive metaphases and only 1/5 patients had detectable Ph-negative metaphases in the CD34+/CD38-subpopulation. We conclude that expression of HLA-DR antigens may precede the expression of CD38 on CD34+ cells during normal stem cell differentiation. In CML DR may be expressed aberrantly and Ph-negative cells are found predominantly in the DR negative subpopulation.     

Cytotoxic activity of platelets and peripheral blood mononuclear cells from oncology patients and healthy donors

Testicular germ cell tumour is said to be a model for curable neoplasm. However, the prognosis of primary extragonadal germ cell tumour does not appear to be as promising. Though similar in histology, the biology of primary extragonadal germ cell tumour is different as exemplified by the patients in this review. Eight patients with primary mediastinal germ cell tumours were treated with intensive cisplatin-based chemotherapy. All, except one, had non-seminomatous components. The poor prognosis of mediastinal germ cell tumour is due to a combination of poor treatment results with the cisplatin-based regimen and the development of non-germ cell and haematological malignancies.