AIDS in a long-term HIV-1-infected patient with a stable high CD4+ cell count and conserved CD4+CD45RO+ subpopulation.
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
C Negri
Publications and source records attributed to C Negri.
Insulin infusion amplifies 17 alpha-hydroxycorticosteroid intermediates response to adrenocorticotropin in hyperandrogenic women: apparent relative impairment of 17,20-lyase activity.
Recent data suggest that insulin is a modulator of ovarian and adrenal steroidogenesis and that, in the ovary of hyperandrogenic women, hyperinsulinemia might cause dysregulation of cytochrome P450c17 alpha activity. To further assess in vivo the effects of insulin on adrenal steroidogenesis, ACTH stimulation was carried out in 21 hyperandrogenic women during a 3-h hyperinsulinemic (80 mU/m2-min) euglycemic clamp. In all of these women the procedure was repeated during saline infusion as n control. In nonamenorrheic patients, the tests were performed in the early follicular phase of two different menstrual cycles. Serum cortisol, progesterone, 17-hydroxypregnenolone (17-OHJPREG). 17-hydroxyprogesterone (17-OHP), dehydroepiandrosterone (DHEA), and androstenedione (A) were measured after 2 h of insulin or saline infusion (zero time) and, subsequently, 30 and 60 min after an iv bolus of 0.25 mg ACTH-(1-24). At zero time, no difference was found in the serum steroid concentrations between the two protocols. ACTH-stimulated serum 17-OHPREG and, to a lesser extent, 17-OHP were significantly higher during insulin than during saline infusion (peaks, 60.6 +/- 9.0 vs. 40.7 +/- 7.9 and 7.7 +/- 7.7 vs. 6.6 +/- 0.6 nmol/L; P < 0.005 and P < 0.01, respectively). Serum DHEA was also slightly higher during hyperinsulinemia, although only after 30 min (54.5 +/- 3.0 vs. 48.2 +/- 4.2 nmol/L; P < 0.05). No statistically significant difference in the cortisol, progesterone, or androstenedione response to ACTH was found between the two protocols. ACTH-stimulated 17-OHPREG/DHEA and 17-OHP/A molar ratios, indexes of apparent 17,20-lyase activity, were significantly higher during the clamp studies than during saline infusion (by ANOVA, F = 12.8; P < 0.001 and F = 6.7; P < 0.005, respectively), suggesting an impaired enzyme activity. These in vivo data support the hypothesis that insulin potentiates ACTH-stimulated steroidogenesis. This effect of insulin seems to be associated with a relative impairment of 17,20-lyase activity.
17-Hydroxycorticosteroids↗
The insulin resistance in women with hyperandrogenism is partially reversed by antiandrogen treatment: evidence that androgens impair insulin action in women.
To assess whether androgen excess per se might impair insulin action, insulin sensitivity was measured by a two-step (20 and 80 mU/m2.min) hyperinsulinemic euglycemic clamp combined with indirect calorimetry and tracer glucose infusion in 43 women (13 obese and 30 nonobese) with normal glucose tolerance and clinical evidence of increased androgen action (hirsutism and/or polycystic ovary syndrome) as well as 12 age- and body mass index-matched healthy controls. Hyperandrogenic women were studied basally and after 3-4 months of antiandrogen treatment with 3 different drugs: spironolactone (n = 23), flutamide (n = 10), or the GnRH agonist buserelin (n = 10). Six women given spironolactone were also reexamined after 1 yr of therapy. At baseline, insulin-mediated glucose uptake was lower in hyperandrogenic women than in controls (by ANOVA, F = 14.3; P < 0.001). Insulin resistance was observed in both ovarian and nonovarian hyperandrogenism, as distinguished by acute GnRH agonist testing. After antiandrogen therapy, insulin action on glucose metabolism significantly increased for both the patients as a whole (F = 7.4; P < 0.01) and each treatment group separately. However, insulin action remained lower than in controls and showed no further improvement in patients reevaluated after I yr of treatment. Increases in both oxidative and nonoxidative glucose metabolism accounted for the improvement in substrate disposal induced by antiandrogen drugs. The increase in the effectiveness of insulin was greater in the lean subjects, whereas the change was small and not statistically significant in the obese women. Response to treatment was more pronounced in women with nonovarian hyperandrogenism, particularly at the low insulin infusion rate. Endogenous glucose production in hyperandrogenic patients was similar to that in healthy women and was unaffected by therapy. In conclusion, antiandrogen treatment partially reversed the peripheral insulin resistance associated with hyperandrogenism regardless of which antiandrogen was used. These data strongly suggest that in women, androgen excess per se contributes to impairment of insulin action.
Adult↗
Sequence of events leading to apoptosis in long term cultured HeLa cells.
We have maintained HeLa cells in culture in the original medium for increasing times to induce growth arrest. Cell viability was evaluated by trypan blue dye exclusion assay. We observed that when cells are maintained in culture for several days, morphological hallmarks of apoptosis become evident. DNA synthesis rate, followed by (3)H-thymidine incorporation slowed down in long term cultured cells. This evidence was supported by the analysis of cell cycle progression determined by proliferating cell nuclear antigen (PCNA) immunostaining. Apoptotic cells have been characterized with respect to the sequential appearance of high molecular weight and internucleosomal DNA fragmentation. We have provided evidence that in this experimental model the first step in DNA degradation is represented by the formation of high molecular weight fragments, whereas nucleosomal DNA ladder is visible later on. The activation of the enzyme poly(ADP-ribose)polymerase, considered a marker of apoptotic death, has been observed. The results suggest that long term culture conditions activate the apoptotic programme.
Journal Article↗
Immunocytometric determination of the alpha and beta isoforms of human DNA topoisomerase II: influence of different fixatives.
The immunoreactivity of the alpha and beta isoforms (170 and 180 kDa, respectively) of human DNA topoisomerase II (topo II) toward specific monoclonal antibodies (MoAbs), was investigated in HeLa cells. The extent of antigen preservation achieved with the dehydrating fixatives, ethanol and acetone, was compared with that obtained with the cross-linking agent paraformaldehyde. Binding of each MoAb to the relative isozyme was detected by indirect immunofluorescence labelling and quantified by flow cytometry. The amount of antigen detectable was calculated by normalizing the mean fluorescence intensity of samples incubated with specific MoAbs, to the respective background fluorescence. For both isozymes, acetone provided the highest immunofluorescence/background ratio. A strong reduction in the immunoreactivity of the 180-kDa isoform was observed after ethanol, and to a minor extent after paraformaldehyde fixation, while reactivity of the 170-kDa isoform was not significantly affected. Cell cycle distribution of each protein was assessed by dual-parameter analysis of immunofluorescence vs. DNA content. No evident difference in the cell cycle distribution of each isozyme was found with each fixation protocol. At the morphological level, the pattern of immunofluorescence showed that the localization of each isozyme was very similar for all these fixatives. These results confirm the great sensitivity of the 180-kDa isoform to degradation, and indicate that fixation has to be carefully chosen in order to determine the relative amount of topo II isoforms with immunocytometric techniques.
Animals↗
Induction of apoptotic cell death by DNA topoisomerase II inhibitors.
We have analyzed the interference of antitumoral drugs acting through the inhibition of DNA topoisomerase II on the human HeLa cell metabolism. Different compounds characterized by a diverse mechanism of action have been used, namely m-amsacrine, an intercalative drug, etoposide, which does not intercalate DNA, and suramin, which exerts its effect through an unknown mechanism. In HeLa cells treated with increasing doses of these drugs, we have examined cell viability and DNA synthesis capacity, and we have evaluated topoisomerase II activity. Cellular morphology and DNA integrity have been studied in order to characterize the mechanism of cell death. The results we have obtained clearly indicate that topoisomerase II poisons induce cell death by apoptosis. These observations suggest a role of the inhibition of topoisomerase II activity in the apoptotic program.
Amsacrine↗
Activation of poly(ADP-ribose)polymerase in apoptotic human cells.
We have studied the effect of the chemotherapeutic drug VP-16 (etoposide) on the metabolism of HeLa cells by analysing different cellular parameters considered as markers of apoptosis. Typical features such as chromatin condensation and internucleosomal DNA cleavage are visible in HeLa cells exposed to VP-16. We investigated whether the appearance of small-sized DNA fragments could regulate the ADP-ribosylation process. To this purpose, we have analysed, by means of the activity gel technique; the structural and catalytical properties of poly(ADP-ribose)polymerase. In extracts from cells where etoposide-induced DNA fragmentation occurred, we have shown that the label of the autoribosylated form of the enzyme is greatly increased even if the amount of the protein remains constant. This phenomenon is completely abolished in cells preincubated with poly(ADP-ribose)polymerase inhibitor, 3-aminobenzamide. After VP-16 administration, we have observed that the level of NAD is not heavily decreased. It is widely agreed that zinc exerts an inhibitory effect on the endonuclease(s) responsible for the fragmentation of DNA during apoptosis. After incubation of cells with zinc/VP-16 we have found the occurrence of apoptotic parameters even in the absence of internucleosomal DNA cleavage. The inhibition of DNA fragmentation prevents the activation of poly(ADP-ribose)polymerase activity. These results indicate that the activation of the enzyme towards the automodification reaction is strictly dependent on the appearance of DNA internucleosomal fragments and could represent a way to control enzyme activity.
Adenosine Monophosphate↗
Flutamide in the treatment of hirsutism: long-term clinical effects, endocrine changes, and androgen receptor behavior.
OBJECTIVE: To investigate the long-term effects of treatment with low doses of flutamide on clinical and hormonal parameters, as well as on the androgen receptor status, in hirsute women. DESIGN: Eighteen hirsute patients with regular menses were studied basally and during treatment with 125 mg flutamide, three times per day for 12 months. Barrier or intrauterine contraception was used during the study in sexually active women. Safety parameters were assessed throughout the study. Hirsutism, graded by the modified Ferriman-Gallwey score, and hormonal parameters were evaluated basally and at 4-month intervals during treatment. Gonadotropin-releasing hormone and ACTH stimulation tests were performed before and after 3 to 4 months of therapy. In addition, the concentration of androgen receptors in mononuclear leukocytes was measured, in both the follicular and luteal phases of the menstrual cycle, basally and after 4 months of flutamide treatment. RESULTS: Flutamide was well tolerated in all women, with the noticeable exception of one patient who presented increased serum transaminase after 8 months of therapy. Hirsutism markedly improved in all women during the treatment (Ferriman-Gallwey score after 1 year: 4.1 +/- 0.5 versus 14.1 +/- 0.9). A reduction of serum androgens was found, whereas no change was observed in either basal or GnRH-stimulated gonadotropins or in the cortisol and 17 alpha-hydroxyprogesterone response to ACTH. Cycles remained ovulatory. Before treatment, the number of androgen receptors was higher in the luteal than in the follicular phase. This rhythmic differentiation disappeared after the patients had been given the antiandrogen drug. CONCLUSIONS: Flutamide is effective in the treatment of hirsutism but requires constant surveillance of liver function. Androgen receptor blockade might be potentiated by a reduction of serum androgens. Flutamide affects androgen receptor behavior during the menstrual cycle. The meaning of this finding remains to be elucidated.
Acne Vulgaris↗
Sustained therapy with 3-hydroxy-3-methylglutaryl-coenzyme-A reductase inhibitors does not impair steroidogenesis by adrenals and gonads.
Plasma lipoproteins are a major source of cholesterol for steroid hormone synthesis. 3-Hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) reductase inhibitors, which reduce both intracellular cholesterol synthesis and serum cholesterol levels, thus have a potential negative impact on steroidogenesis. In this study, we evaluated basal and maximally stimulated adrenocortical and testicular steroidogenesis in 24 hypercholesterolemic male subjects during 6-36 months of statin treatment. One group was evaluated before treatment and after 6 months of treatment. A second group, which received long term treatment, was evaluated after 24-36 months and then 2 months after treatment had been discontinued. Fourteen subjects were given simvastatin, and 12 were given pravastatin, both at the maximum therapeutic dosage of 40 mg/day. During statin therapy, serum cholesterol was lowered by about 30%. Basal serum and urinary cortisol levels as well as serum cortisol response to ACTH were not influenced by statin therapy. Basal serum testosterone and its response to hCG were also unchanged by statin treatment. In addition, steroid hormone urinary metabolites were strikingly similar when patients were given HMG-CoA reductase inhibitors and when they were not. These results indicate that maximum therapeutic doses of statins have no negative impact on adrenocortical and testicular steroidogenesis even when these glands are maximally stimulated.
Adolescent↗
Discrete localization of different DNA topoisomerases in HeLa and K562 cell nuclei and subnuclear fractions.
Monoclonal antibodies raised against DNA topoisomerase I and against topoisomerase II alpha and beta isoforms, which have been previously demonstrated to be highly specific and capable of detecting cell cycle-related variations of the topoisomerase II isoforms (Negri et al., 1992, Exp. Cell Res. 200, 452-459), have been utilized for a fine subcellular localization. Immunocytochemistry by confocal and electron microscopy have been used for a topological and quantitative evaluation of the fine distribution of the different topoisomerases in HeLa and K562 cells. Topoisomerase I and topoisomerase II alpha are present both in the nucleoplasm and in the nucleolus, though at different relative ratios, while topoisomerase II beta is exclusively present at the nucleolar level. This is further confirmed by immunoblotting and immunocytochemical quantitative evaluations performed on purified nuclear matrix fractions obtained from K562 cells. In fact, the amount of topoisomerase I and topoisomerase II alpha present in the whole cell nuclei is partly lost in isolated nuclei but, while topoisomerase I is further significantly reduced in nuclear matrix preparations, the topoisomerase II alpha content is only slightly decreased. On the other hand, the great majority of topoisomerase II beta is retained in the nuclear matrix and can be detected exclusively in association with the nucleolar remnant. These results are consistent with specific functional roles hypothesized for the different topoisomerase types.
Antibodies, Monoclonal↗
Expression of the 170-kDa and 180-kDa isoforms of DNA topoisomerase II in resting and proliferating human lymphocytes.
The expression of the 170-kDa (alpha) and the 180-kDa (beta) isoforms of DNA topoisomerase II (topo II) was investigated with two specific monoclonal antibodies (MoAbs) in human peripheral blood lymphocytes (PBL), before and after phytohaemoagglutinin (PHA) stimulation. Binding of each MoAb was detected by indirect immunofluorescence labelling and quantified with flow cytometry. In resting PBL, the intensity of immunostaining was very low for both isozymes; however, topo IIbeta-associated immunofluorescence was about 2.5 times significantly higher (P<0.001) than that associated with the alpha isoform. Between 48 and 72 h of PHA stimulation, when the highest percentage of cells in S and G2+M phases was found, the levels of topo IIalpha and beta increased up to about 30 and 10 times the value measured in resting PBL, respectively. Thus, the two isoforms reached comparable immunofluorescence values. At longer stimulation periods (96-120 h), topo IIalpha immunofluorescence was not significantly changed, while that relative to topo IIbeta declined to about 50% of the peak value (P<0.02). At this time however, topo IIalpha-associated immunofluorescence was not significantly different from that related to the beta isozyme. These results suggest that in resting PBL topo IIalpha is required at levels lower than topo IIbeta, while in proliferating lymphocytes both isoforms are expressed to significantly higher levels.
Cell Cycle↗
Clinical and hormonal effects of the 5 alpha-reductase inhibitor finasteride in idiopathic hirsutism.
Hyperactivity of 5 alpha-reductase in the skin is considered a major mechanism of excessive hair growth in hirsute women with normal levels of serum androgens (idiopathic hirsutism). Preventing the conversion of testosterone to dihydrotestosterone by inhibiting 5 alpha-reductase activity could thus be the most rational and effective treatment in this condition. The present study evaluated the effects of the oral administration of finasteride (5 mg once daily) for 6 months in 17 young women with idiopathic hirsutism, 5 of whom were also given an oral contraceptive. The degree of hirsutism (graded by a modified Ferriman-Gallwey score), serum sex hormone levels, and serum and urinary 5 alpha-metabolism steroid profiles were determined basally and periodically during the treatment period. The modified Ferriman-Gallwey score showed a remarkable reduction after 6 months of finasteride treatment (5.9 +/- 0.6 vs. 11.7 +/- 1.3; P < 0.01). Serum 5 alpha-dihydrotestosterone and 3 alpha-androstanediol glucuronide levels were decreased, and urinary C19 and C21 5 beta/5 alpha metabolite ratios were increased compared with pretreatment values. No significant adverse effect was reported. In women treated with finasteride and oral contraceptive, clinical efficacy was slightly more pronounced. In conclusion, the 5 alpha-reductase inhibitor finasteride is well tolerated and seems to be a useful tool in the treatment of idiopathic hirsutism.
Adult↗
Dose-dependent zinc inhibition of DNA ladder in apoptotic HeLa cells regulates the activity of poly(ADP-ribose) polymerase and does not protect from death induced by VP-16.
Zinc ions exert an inhibitory effect on Ca(2+)Mg(2+)-dependent endonuclease which is supposed to be responsible for the fragmentation of DNA during apoptosis. In the experimental system we used, that is HeLa cells treated with VP-16, the protection from internucleosomal DNA degradation is modulated by Zn concentration and appears to be dependent on the time after treatment. This effect does not prevent cell death or occurrence of apoptotic parameters, suggesting that DNA ladder appearance is not a crucial event in apoptosis. The activation of poly(ADP-ribose)polymerase following the administration of VP-16, is not observed in cells in which DNA fragmentation has been abolished by zinc, supporting the hypothesis that this event is regulated by the appearance of small-sized DNA fragments.
Journal Article↗
DNA topoisomerase II beta: stability and distribution in different animal cells in comparison to DNA topoisomerase I and II alpha.
DNA topoisomerase I and the isoforms alpha and beta of DNA topoisomerase II were analyzed in different animal cells using a panel of monoclonal antibodies (MoAbs). The beta isoform is a most unstable enzyme. We investigated conditions to stabilize beta isoform because its variability changes according to the derivation of cells. We describe two MoAbs specific to DNA topoisomerase I: the first one recognizes the enzyme in all the species tested including fish; the second one, in contrast, recognizes an epitope present only in mammalian cells. We also found that eight of eight MoAbs against DNA topoisomerase II alpha and five of six against the beta isoform recognize the respective enzymes in all the species tested excluding fish. In addition, MoAbs to the alpha isoform are specific to epitopes not present in the carboxyl third of the enzyme.
Animals↗
ADP-ribosylation of nonhistone proteins in HeLa cells: modification of DNA topoisomerase II.
We have studied the nonhistone proteins which are modified by ADP-ribosylation in HeLa cells. When isolated nuclei were incubated with 32P-NAD, the main labeled proteins presented sizes of 170, 116, 70, and 45 kDa. To provide evidence for the identification of the 170-kDa band as DNA topoisomerase II, the enzyme was immunoprecipitated from isolated nuclei incubated with 32P-NAD and a labeled peptide of 170 kDa was observed. The label was sensitive to the action of venom phosphodiesterase which specifically degrades ADP-ribose. ADP-ribosylated proteins were also isolated from HeLa cells by affinity chromatography on boronate-agarose gel. Using a monoclonal antibody against the 170-kDa isoform of topoisomerase II, a single 170-kDa immunoreactive peptide was recognized by Western blot among the retained protein acceptors. When ADP-ribosylation was blocked by treating HeLa cells with 3-aminobenzamide, topoisomerase II was no longer retained on the boronate column. These results provide experimental evidence indicating that DNA topoisomerase II is ADP-ribosylated in HeLa cells. To possibly correlate ADP-ribosylation of nuclear proteins with the extent of DNA damage, permeabilized HeLa cells were incubated with 32P-NAD after treatment with the alkylating agent dimethylsulfate. ADP-ribosylated proteins were isolated by boronate chromatography. A strong increase in the ADP-ribosylation of the poly(ADP-ribose)polymerase was observed, whereas no further modification of topoisomerase II was noted.
Adenosine Diphosphate Ribose↗
Antibodies to topoisomerase II in idiopathic pulmonary fibrosis.
Antibodies to DNA topoisomerase II (anti-topoisomerase II) were detected by ELISA in the sera of 18 out of 41 (44%) patients with idiopathic pulmonary fibrosis (IPF). Follow-up sera were also obtained from 19 of the patients. DNA topoisomerase II binding remained constantly high or low in the majority of follow-up sera, but 2 out of the 8 positive cases became negative while 3 out of the 11 negative cases became positive during follow-up. No association was found between occurrence of anti-topoisomerase II antibodies and any indices of disease severity. Furthermore, individual patient follow-up did not show any correlation between changes in topoisomerase II binding and deterioration or improvement of clinical status. In conclusion our study shows that although anti-topoisomerase II are detectable in a large fraction (approximately 50%) of IPF patients and are useful for diagnostic purposes, they do not provide a measure of clinical activity.
Adult↗
The effect of the chemotherapeutic drug VP-16 on poly(ADP-ribosylation) in apoptotic HeLa cells.
We have studied the effect of the chemotherapeutic drug VP-16 (etoposide) on the metabolism of HeLa cells by analysing different cellular parameters; in particular we have focused on changes in cellular morphology that are considered as markers of apoptosis. By immunofluorescence experiments we have shown that VP-16 causes the complete disruption of nucleoli and induces chromatin margination and fragmentation. Agarose gel electrophoresis of DNA from cells treated with 10-100 microM VP-16 showed the appearance of a characteristic ladder due to the internucleosomal DNA cleavage. The effect of etoposide on DNA integrity was not prevented by preincubation of cells with the protein synthesis inhibitor cycloheximide. These results provide experimental evidence indicating that the typical features of apoptosis are visible in HeLa cells exposed to VP-16. In this experimental system we have investigated whether the ADP-ribosylation process could be regulated by the presence of DNA fragments. By means of the activity gel technique, which allows the direct evaluation of automodified poly(ADP-ribose)polymerase, we have observed that in extracts from cells where etoposide-induced DNA fragmentation occurred, the autoribosylated form of the enzyme is greatly increased. Ribosylated poly(ADP-ribose)polymerase has been isolated by affinity chromatography on boronate column from cells permeabilized and labelled with [32P]NAD. Drug exposure caused a strong augmentation of modified enzyme. These observations suggest that activation of ADP-ribosylation process occurs in cells that show the typical features of apoptosis.
Apoptosis↗
Detection of circulating autoantibodies to poly(ADP-ribose)polymerase in autoimmune diseases.
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Autoantibodies↗