Pantetheinase activity and cysteamine content in cystinotic and normal fibroblasts and leukocytes

Cysteamine is the most effective agent known for the reduction of the elevated cystine content of cells from patients with cystinosis. A defect in endogenous cysteamine generation could account for many of the metabolic features of this disorder. To test this hypothesis, we have developed improved methods for measuring pantetheinase (cysteamine-generating) activity and intracellular cysteamine levels and used these methods to measure such parameters in cystinotic and normal leukocytes and cultured skin fibroblasts. Pantetheinase activity as defined in the test was similar in extracts of cystinotic and normal cells [leucocytes, normal, 78 +/- 15 (S.E.), cystinotic, 56+/- 6.4; fibroblasts, normal, 9.4 +/- 1.5; cystinotic, 7.7 +/- 1.7]. Cysteamine levels were normal in leukocytes from cystinotics receiving no cysteamine or doses of oral cysteamine too low to reduce leukocyte cystine content. The results indicate that the cause of cystinosis is unlikely to be related to a failure to generate of sustain normal intracellular cysteamine levels.     

Mutation rate of a microsatellite sequence in normal human fibroblasts

Dinucleotide repeats, because of their repetitive nature, are prone to frameshift mutations, most likely via a DNA-polymerase slippage mechanism. Mutation rates in microsatellite DNA sequences are high in mismatch repair-defective cells. In normal cells, only estimates of maximal rates of mutation in microsatellites have been possible previously, because of the low sensitivity of screening assays for mutations in endogenous sequences. We have measured the spontaneous mutation rate of a dinucleotide repeat in diploid human foreskin fibroblasts. In our system, the mutation target is a (CA)17 repeat contained within a stably integrated plasmid. The repeat disrupts the reading frame of a neomycin (neo) resistance gene within the plasmid. Cells containing frameshift mutations in the CA repeat that correct the reading frame of the neo gene are selected using the neo analogue G418. This system of measuring microsatellite mutation rates is highly sensitive, because there is a specific target within which mutations can be selected. Fluctuation analysis of cells containing the target DNA yielded mutation rates of <3.1 x 10(-8) to 44.8 x 10(-8) mutations/cell/generation. This is the first report of a direct measurement of a spontaneous mutation rate of a microsatellite sequence in normal human cells.     

DNA topoisomerase inhibitors induce reversible senescence in normal human fibroblasts

Inhibitors of DNA topoisomerases I and II induced arrest in cell division in normal human fibroblasts depending on cell divisions. Arrested cells showed morphology similar to those of normally senesced cells and strongly induced senescence-associated beta-galactosidase. In these cells, p16ink4a was upregulated, whereas p21waf1 or p53 was not altered. Upon removal of the inhibitors, the cells resumed growth but their cumulative population doublings were reduced dose dependently. Accelerated telomere shortening was not observed in the arrested cells. These results suggest that DNA topoisomerase inhibitors are efficient and reversible inducers of premature senescence in normal human cells.     

Coordinated induction of MRP/GS-X pump and gamma-glutamylcysteine synthetase by heavy metals in human leukemia cells

We recently reported that GS-X pump activity, as assessed by ATP-dependent transport of the glutathione-platinum complex and leukotriene C4, and intracellular glutathione (GSH) levels were remarkably enhanced in cis-diamminedichloroplatinum(II) (cisplatin)-resistant human leukemia HL-60 cells (Ishikawa, T., Wright, C. D., and Ishizuka, H. (1994) J. Biol. Chem. 269, 29085-29093). Now, using Northern hybridization and RNase protection assay, we provide evidence that the multidrug resistance-associated protein (MRP) gene, which encodes a human GS-X pump, is expressed at higher levels in cisplatin-resistant (HL-60/R-CP) cells than in sensitive cells, whereas amplification of the MRP gene is not detected by Southern hybridization. Culturing HL-60/R-CP cells in cisplatin-free medium resulted in reduced MRP mRNA levels, but these levels could be induced to rise within 30 h by cisplatin and heavy metals such as arsenite, cadmium, and zinc. The increased levels of MRP mRNA were closely related with enhanced activities of ATP-dependent transport of leukotriene C4 (LTC4) in plasma membrane vesicles. The glutathione-platinum (GS-Pt) complex, but not cisplatin, inhibited ATP-dependent LTC4 transport, suggesting that the MRP/GS-X pump transports both LTC4 and the GS-Pt complex. Expression of gamma-glutamylcysteine synthetase in the cisplatin-resistant cells was also co-induced within 24 h in response to cisplatin exposure, resulting in a significant increase in cellular GSH level. The resistant cells exposed to cisplatin were cross-resistant to melphalan, chlorambucil, arsenite, and cadmium. These observations suggest that elevated expression of the MRP/GS-X pump and increased GSH biosynthesis together may be important factors in the cellular metabolism and disposition of cisplatin, alkylating agents, and heavy metals.     

Altered protein metabolism in arrested populations of aging human diploid fibroblasts

Protein synthesis and turnover were measured in human diploid fibroblasts which were arrested in an essentially nonmitotic state by reducing the serum concentration in the incubation medium to 0.5%. Through the first 4 days of the arrested period both early and late passage cells lost about 20% of their cellular protein. There was a reduction in the rate of protein synthesis at both passage levels during this period, but there was no significant age-related difference in the synthetic rate or the rate of protein turnover. After day 4 both early and late passage cells maintained a constant protein content, but late passage cells did this while processing more protein through faster rates of both synthesis and turnover than did early passage cells. These results support those theories of cellular senescence which predict altered protein metabolism as a major consequence of the aging process.     

Changes in GSH-antioxidant system induced by daunorubicin in human normal and diabetic fibroblasts

We investigated the effect of daunorubicin on glutathione content and activity of GSH-related enzymes in cultured normal and diabetic human fibroblasts. Cells were incubated with 4 microM daunorubicin (DNR) for 2 h followed by culture in drug-free medium for up to 72 h. Treatment of diabetic cells with the drug caused a time-dependent depletion of intracellular GSH and a decrease of the GSH to total glutathione ratio. GSH depletion was accompanied by apoptotic changes in morphology of the nucleus. Analysis of GSH-related enzymes showed a significant increase of the activities of Se-dependent and Se-independent peroxidases and glutathione S-transferase. In contrast, glutathione reductase activity was reduced by 50%. Significant differences between normal and diabetic cells exposed to DNR were observed in the level of GST and Se-dependent glutathione peroxidase activities. These findings indicated that daunorubicin efficiently affects the GSH antioxidant defense system both in normal and diabetic fibroblasts leading to disturbances in glutathione content as well as in the activity of GSH-related enzymes.     

125I-labeled human epidermal growth factor. Binding, internalization, and degradation in human fibroblasts

125I-labeled human epidermal growth factor (hEGF) binds in a specific and saturable manner to human fibroblasts. At 37 degrees C, the cell-bound 125I-hEGF initially may be recovered in a native form by acid extraction; upon subsequent incubation, the cell-bound 125I-hEGF is degraded very rapidly, with the appearance in the medium of 125I-monoiodotyrosine. At 0 degrees C, cell-bound 125I-hEGF is not degraded but slowly dissociates from the cell. The data are consistent with a mechanism in which 125I-hEGF initially is bound to the cell surface and subsequently is internlized before degradation. The degradation is blocked by inhibitors of metabolic energy production (azide, cyanide, dinitrophenol), some protease inhibitors (Tos-Lys-CH2Cl, benzyl guanidobenzoate), a lysosomotropic agent (chloroquine) various local anesthetics (cocaine, lidocaine, procaine), and ammonium chloride. After the binding and degradation of 125I-hEGF the fibroblasts are no longer able to rebind fresh hormone. The binding capacity of these cells is restored by incubation in a serum-containing medium; this restoration is inhibited by cycloheximide or actinomycin D.     

Hyperphenylalaninemia due to dihydropteridine reductase deficiency. Assay of the enzyme in fibroblasts from affected infants, heterozygotes, and in normal amniotic fluid cells

Two infant siblings with modest elevations of serum phenylalanine concentrations had seizures and developmental regression: they died in their second year. Dihydropteridine reductase activity, which can be measured in normal cultured skin fibroblasts, was measured in the younger sibling and was absent. Parents of the two siblings and parents of a previously reported patient all showed 50% or less of the normal dihydropteridine reductase activity in their cultured fibroblasts. Dihydropteridine reductase activity is also present in normal cultured amniotic fluid cells, offering the possibility of prenatal diagnosis. Absence of dihydropteridine reductase results not only in a defect in the conversion of phenylalaning to tyrosine, but also in the biosynthesis of the neurotransmitters, dopamine, norephinephrine, and serotonin. Since deficiencies in these neurotransmitters would not be alleviated by a phenylalanine-restricted diet, it is important to establish the nature of the enzymatic defect in all suspected variants of phenylketonuria.     

Increase in telomere sequence-binding activity in normal human fibroblasts in senescence or in cells treated with phorbol ester or N-methyl-N'-nitro-N-nitrosoguanidine

The telomere is a specialized chromatin structure composed of unique repetitive DNA sequences and specific nuclear proteins. Telomere sequence-binding activity was measured by a mobility shift assay using nuclear extract from normal human fibroblasts. The specific binding activity to the telomere sequence increased in cells that were in a senescence state compared to that in cells at early population doublings. Treatment of cells with tumor promoting phorbol ester TPA induced an increase in the telomere sequence binding activity of nuclear extract in young cells, but the increase was marginal in senescent cells. DNA-damaging N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) also increased the telomere sequence binding activity in young cells, but not in senescent cells. As a reference, we measured the binding activity to NFkB sequence. It was activated by TPA or okadaic acid, but was not affected by MNNG or in senescence. The increase in telomere sequence-binding activity seemed to depend on activation of tyrosine phosphorylation, since an inhibitor of Tyr-kinase abolished the increase in telomere-binding activity. The molecular weight of the major binding factor in the normal human fibroblasts was approximately 32 kDa which is different from that of the telomere-associated protein, TRF-1.     

Detection of normal and chimeric nucleophosmin in human cells

In anaplastic large-cell lymphoma (ALCL), the (2;5) chromosomal translocation creates a fusion gene encoding the 80-kD NPM-ALK hybrid protein. This report describes three new monoclonal antibodies, two of which recognize, by Western blotting, the N-terminal portion of NPM present in the NPM-ALK fusion protein and also in two other NPM fusion proteins (NPM-RARalpha and NPM-MLF1). The third antibody recognizes the C-terminal portion (deleted in NPM-ALK) and reacts only with wild-type NPM. The three antibodies immunostain wild-type NPM (in paraffin-embedded normal tissue samples) in cell nuclei and in the cytoplasm of mitotic cells. Cerebral neurones, exceptionally, show diffuse cytoplasmic labeling. In contrast to normal tissues, the two antibodies against the N-terminal portion of NPM labeled the cytoplasm of neoplastic cells, in four ALK-positive ALCL, reflecting their reactivity with NPM-ALK fusion protein, whereas the antibody to the C-terminal NPM epitope labeled only cell nuclei. Immunocytochemical labeling with these antibodies can therefore confirm that an ALK-positive lymphoma expresses NPM-ALK (rather than a variant ALK-fusion protein) and may also provide evidence for chromosomal anomalies involving the NPM gene other than the classical (2;5) translocation.     

Phenotype of low proliferative potential is dominant in hybrids of normal human fibroblasts

A biochemical selection system was used to isolate hybrids after fusion of human diploid fibroblast clones of varying proliferative potential. The distribution of proliferative potentials of the hybrids resembled that of the parent with smaller proliferative potential. Therefore, the phenotype of limited division was dominant in hybrids. This dominance was associated with cells that could achieve seven or less divisions present in the parent populations. Terminally nondividing cells present in clones near the end of their proliferative potential differed from those present in clones that had ceased division (senescent), in that a higher percentage could be induced to divide at least once following fusion with a cell capable of division.     

Recombinant human acid alpha-glucosidase corrects acid alpha-glucosidase-deficient human fibroblasts, quail fibroblasts, and quail myoblasts

Thirty-seven children with intrauterine growth retardation (IUGR) were enrolled in a 3-mo longitudinal study. Weight, length, and knee-heel length (by knemometry) were measured at birth and at 7, 14, 30, 60, and 90 d. GH, IGF-I, IGF binding protein (BP)-3, IGFBP-1, and C-peptide were measured at birth and at 2 mo. IGFBP-3 Western immunoblotting and proteolytic activity assay were also performed. Twenty-five newborns with birth weight appropriate for gestational age were chosen as controls. At birth IUGR newborns showed levels of GH and IGFBP-1 significantly higher, and IGF-I, IGFBP-3, and C-peptide significantly lower than control subjects. At 2 mo GH and IGFBP-1 levels decreased, whereas IGF-I, IGFBP-3, and C-peptide rose, attaining the concentrations found in control subjects at birth. Baseline peptide levels as well as their 2-mo variations did not correlate with the gain in weight, supine length, and knee-heel length recorded at 3 mo. Fourteen of nineteen IUGR cord blood samples showed the presence of the intact approximately 42-39-kD IGFBP-3 doublet and the major approximately 29-kD fragment. At 2 mo the IGFBP-3 band pattern was characterized by the predominance of a approximately 18-kD fragment in 6 of 19 tested IUGR infants. The incubation of 2-mo IUGR samples with normal adult serum induced the appearance of the approximately 18-kD band, which was not modified by the addition of EDTA. These results suggest that: 1) the IGF-related growth-promoting mechanism is impaired in IUGR children at birth but is fully restored at 2 mo; 2) the cord blood levels of GH, IGF-I, IGFBP-3, IGFBP-1, and C-peptide are not predictive of the weight and length gain during the first 3 mo of life; 3) IUGR children have at least two different IGFBP-3 proteases, one cation-dependent protease that is present at birth and able to yield the major approximately 29-kD IGFBP-3 fragment and a second one, with a different activation timing, which exhibits cation independence and induces the formation of a approximately 18-kD IGFBP-3 form.     

Cultures of normal human mammary cells

This paper describes a method of obtaining epithelial cells from large quantities of normal human breast tissue and the response of these cells in culture to lactogenic hormones. Suspensions of single cells and clusters of cells resembling normal ductal and alveolar structures were obtained by mechanical disaggregation and subsequent (3h) incubation of tissue fragments in 0.5 mg/ml collagenase. Cells rapidly attached to glass or plastic surfaces within 48 h and grew to form large colonies which maintained their epithelial appearance throughout 2 months of observation. Cell cycling as monitored by DNA synthesis was enhanced by insulin, hydrocortisone, or ovine prolactin (in concentrations of 5.0mug/ml each) at respectively 2,3 and 5 days of incubation. These results were observed in cultures derived from 3 premenopause samples of mammary tissue maintained in medium with 1% fetal calf serum. Prolactin at a concentration of 5 mug/ml induced phosphoprotein synthesis 8-fold over control values. In addition, prolactin induced morphological changes in cells including the development of distended endoplasmic reticulum, large microvilli, and the deposition of glycogen granules. These initial results led to the tentative conclusion that prolactin was sufficient to initiate some of the characteristics in cultured cells normally associated with lactating tissues.     

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.     

RNA metabolism of the normal and the copper treated human endometrium

Ribonucleic acid (RNA) metabolism of the normal and copper-treated ( Cu-T200 IUD) human endometrium was investigated. The relative concentration of total, messenger, ribosomal and transfer RNA was measured in normal and Cu-treated endometrium using the technique of affinity chromatography in polysepharose. The transition from the proli ferative to the secretory endometrium in normal women was accompanied by significant increases (p less than .05) in total RNA, messenger RNA and in ribosomal RNA. The relative proportions of bound and free messenger RNA were also modified by endometrial maturation changing from 70% bound messenger RNA in the proliferative to 83% in the secretory phase. Cu-T200 Cu release appeared to particularly affect RNA metabolism in the secretory phase. During the proliferative phase only the concentration of transfer RNA and the proportion of bound to free messenger RNA were modified by the Cu-T200. The Cu-T200 induced significant decreases (p less than .01 and p less than .05) in all RNA parameters, with the exception of the RNA/deoxyribonucleic acid ratio.     

Effects and metabolism of steroid hormones in human neuroblastoma cells

The development of the central nervous system is influenced by sex steroids and by their metabolites. However, little information on the possible effects of steroid hormones on neuroblastoma cells is available. Human neuroblastoma cell lines have been used as a model of human neuroblasts in vitro to study the metabolism of steroid hormones; in addition, the effects of steroids and steroid antagonists on neuroblastoma cell growth have also been investigated. The results obtained show that SH-SY5Y human neuroblastoma cells may actively metabolize testosterone and progesterone to their respective 5 alpha-reduced metabolites and that differentiation of neuroblastoma cells is paralleled by a significant increase in expression of the type-1 5 alpha-reductase and of the formation of steroid metabolites. All these data are suggestive of a potential role of steroid 5 alpha-reduced metabolites in the biology of neuroblastoma cells. Studies performed to analyze the role of steroid hormones on neuroblastoma cell proliferation show that progesterone at low doses may induce minor stimulation, and at higher doses, a toxic effect on the neuroblastoma cell line SK-N-SH is seen. Moreover, the antiprogestin 17 beta-hydroxy-11 beta-(4-dimethylamino-phenyl-1)-17-(prop-1-ynyl)estra-4,9-dien+ ++-3-one (RU486) decreases the proliferation of these cells in a dose-dependent manner. The effect of RU486 is not antagonized by either progesterone or dexamethasone, a result that seems to exclude the action of RU486 via classic intracellular steroid hormone receptors.