Health burden of co-morbid asthma and allergic rhinitis in West Indian children.

BACKGROUND: Co-morbid allergic rhinitis (AR) and asthma has not been studied in Caribbean countries where there is a high prevalence of childhood asthma. METHODS: Using the International Primary Care Airways Group (IPAG) guidelines to determine AR, care-givers of 393 (response rate=100%) children attending asthma clinics in selected public sector health facilities in Trinidad, West Indies, were interviewed. RESULTS: Children (393) were between 2-17 years and included 239 (60.8%) boys and 154 (39.2%) girls. As many as 53.9% of children sampled (95% CI 45.9-55.8) suffered from AR. Children exposed to household smoking were nearly twice as likely to have AR (p<0.0041, OR=1.9, CI 1.22-2.88). Significantly (p<0.01) more asthmatics with AR (154, 58.6%) visited Accident and Emergency (A&E) in the past 12 months. The odds of visiting A&E at least once in the past 12 months for asthmatics with AR were 1.75 (95% CI 1.15-2.68). The average frequency of A&E visits was higher in children who also suffered from AR (1.75 vs 1.36, p<0.04). Age was negatively correlated (-0.21, p<0.005) with exacerbation frequency for asthmatics without AR suggesting A&E visits are independent of age in co-morbid disease. More children with AR (>60%) suffer day and night symptoms (p<0.001), and miss school (59.8%) (p<0.03) at least once a week (p<0.002) than asthmatics without AR (OR=1.5, 95% CI=1.03-2.30). CONCLUSIONS: AR is prevalent in 53.9% of Trinidadian children with asthma. The burden of co-morbid disease in asthmatic children is associated with increased likelihood of asthma-related A&E visits, day and night symptoms and absence from school.

Genotoxic stress confers preferential and coordinate messenger RNA stability on the five gadd genes.

The growth arrest and DNA damage-inducible (gadd) genes represent a group of five stress-inducible genes that are coordinately regulated at the transcriptional level. Posttranscriptional regulation of gadd153, gadd45, gadd34, gadd33, and gadd7 was studied after exposure to DNA-damaging agents or other growth arrest treatments in hamster cells. Relative transcript levels were measured following treatment with the transcriptional inhibitor actinomycin D. After exposure to methylmethane sulfonate or UV radiation, all five gadd messages demonstrated a coordinate increase in mRNA stability compared to untreated exponentially growing cells. This enhanced stability was not an universal response to genotoxic stress since other DNA damage-inducible genes, such as c-jun and c-fos, did not show an appreciable increase in mRNA half-life. In contrast, induction of growth arrest by media depletion (starvation) or by treatment with the growth inhibitor prostaglandin A2 did not induce such an increase in mRNA stability in all gadd genes. Comparison of overall RNA turnover by 3H labeling of total cellular RNA also indicated that the preferential stabilization of the gadd transcripts by DNA-damaging agents was not an artifactual response due to variations in overall RNA metabolism within each treatment group. However, DNA-damaging agents were ineffective in inducing stabilization of gadd153 mRNA in growth-arrested cells. This suggest that the signal(s) that give rise to gadd mRNA stability may also be affected by the state of cellular proliferation. Together, these results suggest that the global posttranscriptional response of the gadd genes to DNA-damaging agents is specific and unique to actively growing cells, and further implicates the role of the gadd genes in the DNA damage response of cycling cells.

Correlations between S and G2 arrest and the cytotoxicity of camptothecin in human colon carcinoma cells.

Previous cell line comparisons indicated that neither S-phase fraction nor topoisomerase I (top1) levels are sufficient to predict camptothecin (CPT) cytotoxicity (F. Goldwasser el al., Cancer Res., 55: 2116-2121, 1995.). To identify new determinants for CPT activity, two mutant p53 human colon cancer cell lines, SW620 and KM12, that were previously reported to have similar top1 levels and differential sensitivity to CPT were studied. No difference in the kinetics of top1-mediated DNA single-strand breaks or DNA synthesis inhibition were observed after 1 h exposure to 1 microM CPT. Pulse-labeling alkaline elution showed deficiency of damaged replicon elongation in the more sensitive SW620 cells. Consistentiy, flow cytometry analyses showed that KM12 was arrested in G2, whereas SW620 cells were irreversibly blocked in S phase. Aphidicolin protection was minimal in KM12 and more pronounced in the more sensitive SW620 cells. Thus, CPT appears to have two cytotoxic mechanisms, one protectable by aphidicolin and present in SW620 and the other not protectable by aphidicolin and common to both cell lines. SW620 exhibited also a greater capacity to break through the G2 checkpoint after DNA damage. Consistently, SW620 cells failed to down-regulate cyclin B-cdc2 kinase activity, whereas KM12 cells down-regulated cyclin B/cdc2 kinase activity within 30 min to 20 % of control level after CPT treatment. Analysis of the 7 human colon carcinoma cell lines of the NCI Anticancer Drug Screen showed that defects in replicon elongation and G2 breakthrough capability correlate with sensitivity to CPT. Our results suggest that misrepair of damaged replicons and/or alterations in DNA damage checkpoints is critical to defining chemosensitivity to CPT-induced top1-cleavable complexes and that CPT appears to have two cytotoxic mechanisms, one protectable by aphidicolin, and the other not.

Role of the p53 tumor suppressor gene in cell cycle arrest and radiosensitivity of Burkitt's lymphoma cell lines.

We have assessed the role of the p53 tumor suppressor gene in cell cycle arrest and cytotoxicity of ionizing radiation in 17 Burkitt's lymphoma and lymphoblastoid cell lines. Cell cycle arrest was assessed by flow cytometry of cells 16 h following irradiation. In addition to the usual G2 arrest, the cell lines exhibited three types of responses in G1: Class I, strong arrest in G1 following radiation; Class II, minimal arrest; and Class III, an intermediate response. All Class I cells contained normal p53 genes. Of the ten lines that showed minimal G1 arrest, eight had mutant p53 alleles, and two lines were heterozygous for p53 mutations. Both of the lines showing an intermediate response contained wild-type p53. Our results are consistent with the view that mutations abrogate the ability of p53 to induce G1 arrest following radiation. Studies with the heterozygotes showed that the mutant protein can have a dominant negative influence upon wild-type p53, and the reduced ability of two normal p53 lines to arrest in G1 indicated that p53 function can be impaired by other mechanisms. The radiosensitivity of most of the lines appeared to depend on the ability of p53 to induce a G1 arrest. The mean radiation dose that inhibited proliferation of the Class I lines by 50% was 0.98 Gy. Of the eight p53 mutant cell lines tested, five lines required approximately 2.9 Gy to cause a 50% inhibition of cell proliferation. The two heterozygotes were also more resistant to radiation than the Class I cells (50% inhibitory dose, 2.1 and 2.9 Gy). Our results suggest that radioresistance is afforded by a loss of function of wild-type p53, which would normally induce a G1 arrest and promote cell death in the presence of DNA damage.

Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents.

In the present study, we report the characterization of the p53 tumor suppressor pathway in the 60 cell lines of the National Cancer Institute (NCI) anticancer drug screen, as well as correlations between the integrity of this pathway and the growth-inhibitory potency of 123 anticancer agents in this screen. Assessment of p53 status in these lines was achieved through complete p53 cDNA sequencing, measurement of basal p53 protein levels and functional assessment of (a) transcriptional activity of p53 cDNA from each line in a yeast assay, (b) gamma-ray-induced G1 phase cell cycle arrest, and (c) gamma-ray-induced expression of CIP1/WAF1, GADD45, and MDM2 mRNA. Our investigations revealed that p53 gene mutations were common in the NCI cell screen lines: 39 of 58 cell lines analyzed contained a mutant p53 sequence. cDNA derived from almost all of the mutant p53 cell lines failed to transcriptionally activate a reporter gene in yeast, and the majority of mutant p53 lines studied expressed elevated basal levels of the mutant p53 protein. In contrast to most of the wild-type p53-containing lines, cells containing mutant p53 sequence were also deficient in gamma-ray induction of CIP1/WAF1, GADD45, and MDM2 mRNA and the ability to arrest in G1 following gamma-irradiation. Taken together, these assessments provided indications of the integrity of the p53 pathway in the 60 cell lines of the NCI cell screen. These individual p53 assessments were subsequently used to probe a database of growth-inhibitory potency for 123 "standard agents," which included the majority of clinically approved anticancer drugs. These 123 agents have been tested against these lines on multiple occasions, and a proposed mechanism of drug action had previously been assigned to each agent. Our analysis revealed that cells with mutant p53 sequence tended to exhibit less growth inhibition in this screen than the wild-type p53 cell lines when treated with the majority of clinically used anticancer agents: including DNA cross-linking agents, antimetabolites, and topoisomerase I and II inhibitors. Similar correlations were uncovered when we probed this database using most of the other indices of p53 status we assessed in the lines. Interestingly, a class of agents that differed in this respect was the antimitotic agents. Growth-inhibitory activity of these agents tended, in this assay, to be independent of p53 status. Our characterization of the p53 pathway in the NCI cell screen lines should prove useful to researchers investigating fundamental aspects of p53 biology and pharmacology. This information also allows for the large-scale analysis of the more than 60,000 compounds tested against these lines for novel agents that might exploit defective p53 function as a means of preferential toxicity.

WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis.

The tumor growth suppressor WAF1/CIP1 was recently shown to be induced by p53 and to be a potent inhibitor of cyclin-dependent kinases. In the present studies, we sought to determine the relationship between the expression of WAF1/CIP1 and endogenous regulation of p53 function. WAF1/CIP1 protein was first localized to the nucleus of cells containing wild-type p53 and undergoing G1 arrest. WAF1/CIP1 was induced in wild-type p53-containing cells by exposure to DNA damaging agents, but not in mutant p53-containing cells. The induction of WAF1/CIP1 protein occurred in cells undergoing either p53-associated G1 arrest or apoptosis but not in cells induced to arrest in G1 or to undergo apoptosis through p53-independent mechanisms. DNA damage led to increased levels of WAF1/CIP1 in cyclin E-containing complexes and to an associated decrease in cyclin-dependent kinase activity. These results support the idea that WAF1/CIP1 is a critical downstream effector in the p53-specific pathway of growth control in mammalian cells.

Manipulating walking path configuration influences gait variability and six-minute walk test outcomes in older and younger adults.

This study determined whether manipulations to walking path configuration influenced six-minute walk test (6MWT) outcomes and assessed how gait variability changes over the duration of the 6MWT in different walking path configurations. Healthy older (ODR) and younger (YNG) (n=24) adults completed familiarisation trials and five randomly ordered experimental trials of the 6MWT with walking configurations of; 5, 10 and 15m straight lines, a 6m by 3m rectangle (RECT), and a figure of eight (FIG8). Six-minute walk distance (6MWD) and walking speed (m.s(-1)) were recorded for all trials and the stride count recorded for experimental trials. Reflective markers were attached to the sacrum and feet with kinematic data recorded at 100 Hz by a nine-camera motion capture system for 5m, 15m and FIG8 trials, in order to calculate variability in stride and step length, stride width, stride and step time and double limb support time. Walking speeds and 6MWD were greatest in the 15m and FIG8 experimental trials in both groups (p<0.01). Step length and stride width variability were consistent over the 6MWT duration but greater in the 5m trial vs. the 15m and FIG8 trials (p<0.05). Stride and step time and double limb support time variability all reduced between 10 and 30 strides (p<0.01). Stride and step time variability were greater in the 5m vs. 15m and FIG8 trials (p<0.01). Increasing uninterrupted gait and walking path length results in improved 6MWT outcomes and decreased gait variability in older and younger adults.

Antibacterial and anti-inflammatory agents that target endotoxin.

Antibiotic-resistant bacterial infections are a major clinical problem. Lipid A, the active part of lipopolysaccharide endotoxins in Gram-negative bacteria, is an intriguing target for new antibacterial and anti-inflammatory agents. Inhibition of lipid A biosynthesis kills most Gram-negative bacteria, increases bacterial permeability to antibiotics and decreases endotoxin production.

Beta 1-adrenergic and dopamine (D1)-receptors coupled to adenylyl cyclase activation in GT1 gonadotropin-releasing hormone neurosecretory cells.

Binding sites labeled by the beta-adrenergic receptor radioligand (-)-[125I]iodocyanopindolol ([125I]ICYP) and the selective D1-subtype dopamine (DA) receptor radioligand (+)-[125I]SCH 23982 were identified on immortalized hypothalamic GnRH neurons (GT1-7 cell lines). Saturation analyses in crude particulate suspensions of GT1 cells described high affinity and low capacity binding sites for [125I]ICYP (Kd, 41 pM; binding capacity, 25 fmol/mg protein) and [125I]SCH 23982 (Kd, 320 pM; binding capacity, 23 fmol/mg protein). These binding sites were further characterized in competition assays using a variety of agonists and antagonists selective for either beta-adrenergic or DA receptor subtypes. The pharmacological profiles of [125I]ICYP and [125I]SCH 23982 binding obtained from these studies indicated that the radioligands were labeling beta 1-adrenergic and D1-dopaminergic receptor sites, respectively. Northern blot analyses of purified GT1 cell mRNA documented the expression of D1-dopaminergic and beta 1-adrenergic receptor mRNAs. beta 2-Adrenergic receptor mRNA was not identified. All three transcripts were detected in mouse brain mRNA. Both beta 1-adrenergic and D1-receptors were discovered to be positively coupled to adenylyl cyclase. DA and the beta-adrenergic agonist isoproterenol each provoked a rapid and marked stimulation of adenylyl cyclase activity in GT1 cell membrane suspensions. Subtype-selective beta-adrenergic and DA receptor antagonists were used to inhibit isoproterenol- and DA-stimulated adenylyl cyclase activities. Their relative potencies indicated that the isoproterenol stimulation was mediated via the beta 1-adrenergic receptor. The DA-stimulated adenylyl cyclase activity was mediated via the D1-DA receptor. These studies have identified functional beta 1-adrenergic and D1-dopaminergic receptors positively coupled to adenylyl cyclase on GT1 GnRH neurosecretory cells. The existence of these receptors suggests that the noradrenergic and dopaminergic regulation of gonadotropin secretion may be mediated at least in part via direct synapses on GnRH neurons.

Prevalence and clinical characteristics of a high cardiac output state in patients with multiple myeloma.

PURPOSE: This study was undertaken to define the prevalence and clinical characteristics of patients with a high cardiac output state associated with multiple myeloma. PATIENTS AND METHODS: Specifically, we evaluated clinical, laboratory, and two-dimensional and Doppler echocardiographic data in 36 patients with multiple myeloma. Cardiac output was determined noninvasively by a pulsed Doppler technique. RESULTS: A high cardiac output state, defined as a cardiac index greater than or equal to 4.0 L/minute/m2, was present in eight of 34 (23.5%) subjects in whom cardiac output was measurable. None of the known causes of high output states could be identified in these patients. Four patients developed high output congestive heart failure, two of whom died. Age, sex, degree of anemia, serum calcium level, immunoglobulin type, or disease stage did not differ significantly between subjects with or without high output states. However, severe bone involvement was significantly more frequent in those patients with high cardiac output states, occurring in all eight patients with high cardiac indexes compared with nine of 26 patients with low or normal cardiac indexes (p = 0.001). CONCLUSION: These data demonstrate that high cardiac output states are relatively common in patients with multiple myeloma and are associated with the presence of extensive bone disease.

Outcome after prolonged balloon inflations of greater than 20 minutes for initially unsuccessful percutaneous transluminal coronary angioplasty.

Prolonged balloon inflation with or without autoperfusion techniques is a common initial approach to major dissection or abrupt occlusion after percutaneous transluminal coronary angioplasty (PTCA). To assess such a strategy in the setting of unsuccessful angioplasty, 40 patients who underwent prolonged balloon inflations of greater than 20 minutes between January and July of 1991 after initially unsuccessful angioplasty were studied. These patients (median age 59 years) underwent PTCA for progressive or unstable angina (16[40%]), symptomatic or asymptomatic residual stenosis after myocardial infarction (10[25%]), acute myocardial infarction (3[8%]), stable angina (3[8%]), reinfarction (2[5%]), and other indications (6[15%]). The significant stenoses were primarily in the proximal and midportions of the right coronary (53%), left anterior descending (30%) and left circumflex (17%) coronary arteries. Before prolonged balloon inflation, the longest single inflation was 11 +/- 6 minutes and the total time of all inflations was 17 +/- 8 minutes (mean +/- standard deviation). Stenosis was reduced from 91 +/- 9 to 68 +/- 16% before prolonged inflation. After prolonged balloon inflation of 30 +/- 9 minutes, the residual stenosis was 47 +/- 21% (p = 0.0001 vs value before prolonged inflation). Furthermore, improvements in the appearance of filling defects or dissections, or both, occurred in 19 patients (48%). Procedural success was obtained in 32 of 40 patients (80%). Coronary bypass grafting was performed in 8 patients (20%): 4 after unsuccessful PTCA (3 emergently) and 4 electively after initially successful PTCA. Although 5 patients had creatine kinase-MB elevations greater than 20 IU/liter after the procedure, only 1 sustained a Q-wave myocardial infarction. There were no deaths in the hospital.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of long coronary artery narrowings with long angioplasty balloon catheters.

Balloon angioplasty of long coronary artery narrowings has been associated with a lower rate of acute success, and a higher rate of acute complications and restenosis than that observed for short narrowings. Angioplasty catheters with longer length balloons (30 and 40 mm) are now available, and the objective of this study was to determine the acute and long-term success for patients with long coronary artery narrowings treated with these longer balloons. All patients with long narrowings (> or = 10 mm) treated with long balloons at 1 institution over a 1-year period were identified (93 narrowings in 89 patients), and acute and long-term outcomes were carefully documented. Procedural success (residual stenosis < or = 50%) was 97%. Abrupt closure occurred in 6% and major dissection in 11% of narrowings. Clinical success (procedural success without in-hospital death, bypass surgery or myocardial infarction) was achieved in 90% of patients. Repeat catheterization was performed in 61 patients (76% of those eligible), and restenosis was found in 50 to 55%, depending on the definition used. The treatment of long coronary artery narrowings using angioplasty catheters with longer balloons leads to high rates of acute success. However, there is a high rate of restenosis. New interventional devices for long lesions should be compared with long balloons in a randomized controlled trial.

Comparison of bivalirudin versus heparin during percutaneous coronary intervention (the Randomized Evaluation of PCI Linking Angiomax to Reduced Clinical Events [REPLACE]-1 trial).

To assess the efficacy of the direct thrombin inhibitor bivalirudin relative to heparin during contemporary coronary intervention, 1,056 patients who underwent elective or urgent revascularization were randomized in a large-scale pilot study to receive heparin (70 U/kg initial bolus) or bivalirudin (0.75 mg/kg bolus, 1.75 mg/kg/hour infusion during the procedure). All patients received aspirin; pretreatment with clopidogrel was encouraged, and glycoprotein (GP) IIb/IIIa blockade was at the physician's discretion. Stents were placed in 85% of patients; 72% received a GP IIb/IIIa inhibitor, and 56% were pretreated with clopidogrel. Activated clotting times were higher among patients randomized to bivalirudin than among those given heparin before device activation (median 359 vs 293 seconds, p <0.001). The composite efficacy end point of death, myocardial infarction, or repeat revascularization before hospital discharge or within 48 hours occurred in 5.6% and 6.9% of patients in the bivalirudin and heparin groups, respectively (p = 0.40). Major bleeding occurred in 2.1% versus 2.7% of patients randomized to bivalirudin or heparin, respectively (p = 0.52). This trial represents the largest prospective dataset of bivalirudin administered concomitantly with planned GP IIb/IIIa blockade and provides evidence of the safety and efficacy of this combined antithrombotic approach.

Status of the mdm-2 and waf-1 genes in mouse epidermal jb6 variants harboring wild-type p53 - a p53-independent induction of waf-1.

Mutational inactivation of the p53 tumor suppressor gene has been found not to be involved in preneoplastic-to-neoplastic progression in mouse JB6 variants. To examine the role of an inactivated p53 pathway in this tumor promotion/progression model, we have studied the possible alteration of the MDM-2 oncogene, a gene whose product binds to and inactivates p53, and WAF-1 tumor suppressor gene, a gene transcriptionally controlled by p53 that mediates p53 tumor suppression. Alteration of either of these two genes might mimic p53 inactivation in cells expressing wild-type p53. Northern analysis revealed that MDM-2 expression was, in general, upregulated in neoplastic JB6 cells as compared with preneoplastic cells. This higher expression was not due to the gene amplification. Mutational analysis of WAF-1 revealed a) no point mutation in neoplastic cells; b) two polymorphic sites; and c) three nucleotide disagreements with the published sequence. Expression of the WAF-1 gene was also found to be, in general, higher in neoplastic cells, and induced by TPA and/or TNF-alpha in a p53-independent manner. The overall induced level of WAF-1 mRNA was higher in apoptosis sensitive cells after TPA/TNF-alpha treatment, suggesting a role of WAF-1 in mediating apoptosis. We conclude from this study that a) there is no evidence for mutational inactivation of WAF-1 that might mimic p53 inactivation in the JB6 model; b) elevated expression of MDM-2 and/or WAF-1 might be involved in neoplastic progression; and c) there is a p53-independent pathway controlling WAF-1 expression which may mediate p53-independent apoptosis.

The clinical manifestations of cardiac mucormycosis.

The manifestations of cardiac mucormycosis may dominate the clinical picture of disseminated mucormycosis. These manifestations include myocardial infarction, congestive heart failure, conduction system disease, valvular imcompetence and pericarditis. The development of such manifestations in a febrile compromised host with one or more predisposing factors should prompt consideration of disseminated mucormycosis in the differential diagnosis and initiation of appropriate diagnostic and therapeutic strategies.

Rhodococcus equi pneumonia. An unusual early manifestation of the acquired immunodeficiency syndrome (AIDS).

Infection with Rhodococcus equi has been reported as an occasional cause of cavitary pneumonia in severely immunocompromised patients, including those with the acquired immunodeficiency syndrome (AIDS). We report two cases of R equi pneumonia presenting in one month in patients infected with human immunodeficiency virus (HIV) who had not previously had an opportunistic infection. The clinical and radiographic manifestations of the disease are distinctive and should suggest the diagnosis. R equi pneumonia in a person with HIV infection should be considered diagnostic of AIDS. Recognition of this entity is important since antibiotic therapy is different from that conventionally used in pneumonias in AIDS patients and must be prolonged.

Economic analysis of conventional-dose chemotherapy compared with high-dose chemotherapy plus autologous hematopoietic stem-cell transplantation for metastatic breast cancer.

We performed an economic analysis of data from 180 women in a clinical trial of conventional-dose chemotherapy vs high-dose chemotherapy plus stem-cell transplantation for metastatic breast cancer responding to first-line chemotherapy. Data on resource use, including hospitalizations, medical procedures, medications, and diagnostic tests, were abstracted from subjects' clinical trial records. Resources were valued using the Medicare Fee Schedule for inpatient costs at one academic medical center and average wholesale prices for medications. Monthly costs were calculated and stratified by treatment group and clinical phase. Mean follow-up was 690 days in the transplantation group and 758 days in the conventional-dose chemotherapy group. Subjects in the transplantation group were hospitalized for more days (28.6 vs 17.8, P=0.0041) and incurred higher costs (US dollars 84055 vs US dollars 28169) than subjects receiving conventional-dose chemotherapy, with a mean difference of US dollars 55886 (95% CI, US dollars 47298-US dollars 63666). Sensitivity analyses resulted in cost differences between the treatment groups from US dollars 36528 to US dollars 75531. High-dose chemotherapy plus stem-cell transplantation resulted in substantial additional morbidity and costs at no improvement in survival. Neither the survival results nor the economic findings support the use of this procedure outside of the clinical trial setting.

Genotoxic-stress-response genes and growth-arrest genes. gadd, MyD, and other genes induced by treatments eliciting growth arrest.

As discussed throughout this paper, many mammalian DDI genes are associated with growth responses, including both positive responses to growth stimulation and negative responses involving transient growth arrest and terminal differentiation. It is interesting that several immediate-early genes encoding transcription factors, the jun genes, are DDI, are induced by terminal differentiation, and also are associated with positive growth responses. In negative growth-response genes, their control is complex and almost certainly involves multiple regulatory mechanisms. The role of growth-arrest genes after exposure to DNA-damaging agents is currently not known, but as growth arrest can have a protective effect on cells exposed to DNA-damaging agents in both bacteria and eukaryotes, some protective role(s) for the gadd genes may exist. Whatever the roles are for the individual gadd genes, the response of the gadd genes to DNA-damaging agents and other growth-arrest signals has been highly conserved during mammalian evolution, and it is likely that this stress response, as reflected by induction of one or more gadd genes, is present in most or perhaps all mammalian cells. Our findings that the gadd group overlaps with another group of growth-arrest genes, the MyD, indicate that these two groups combined define a new class of genes whose protein products are likely to play a role in cell growth cessation.

ATP secretion and extracellular protein phosphorylation by CNS neurons in primary culture.

The significant role of secreted ATP in the regulation of neuronal function and the activity of ecto-protein kinases which utilize extracellular ATP to phosphorylate proteins localized at the cell surface have been previously studied in peripheral neurons and in cloned neural cell lines. In the present study we have utilized neostriatal neurons differentiated in primary culture to demonstrate vesicular secretion of ATP and phosphorylation of proteins by extracellular ATP in neurons derived from the central nervous system (CNS). Neostriatal neurons from embryonic mice were maintained in a chemically defined medium for 15-18 days. Functional differentiation was determined by measuring evoked GABA-release. ATP-secretion was measured by luciferin-luciferase assays, and protein phosphorylation by adding gamma-32P-ATP to the extracellular medium. Depolarization by 50 mM KCl induced a Ca++-dependent ATP release, and stimulation by 100 microM veratridine resulted in secretion of ATP that could be blocked by tetrodotoxin. Phosphorylation of specific protein components with apparent molecular mass of 110 Kd, 80 Kd, 55 Kd, 30 Kd and 20 Kd was detected in striatal neurons incubated for 15 min with gamma-32P-ATP added to the medium, but not by labeling intracellular ATP pools with equivalent amounts of radioactivity presented as inorganic 32Pi. These results open for investigation the role of extracellular protein phosphorylation systems in processes underlying the responsiveness of CNS neurons to secreted ATP.

Antiproliferative effects of cyclopentenyl cytosine (NSC 375575) in human glioblastoma cells.

Cyclopentenyl cytosine (CPEC) exerts an antiproliferative effect against a wide variety of human and murine tumor lines, including a panel of human gliosarcoma and astrocytoma lines. This effect is produced primarily by the 5'-triphosphate metabolite CPEC-TP, an inhibitor of cytidine-5'-triphosphate (CTP) synthase (EC 6.3.4.2). Because previous studies with human glioma cell lines utilized cells in long-term tissue culture, we have undertaken to determine whether the activity of CPEC in such model systems is also demonstrable in freshly excised human glioblastoma cells. Glioma cells obtained at surgery and in log phase growth were exposed to the drug at levels ranging from 0.01 to 1 microM for 24 h, and CPEC-TP and CTP levels were determined by HPLC. Dose-dependent accumulation of CPEC-TP was accompanied by a concomitant decrease in CTP pools, with 50% depletion of the latter being achieved at a CPEC level of ca. 0.1 microM. Human glioma cell proliferation was inhibited 50% by 24-h exposure to 0.07 microM CPEC. Postexposure decay of CPEC-TP was slow, with a half-time of 30 h. DNA cytometry showed a dose-dependent shift in cell cycle distribution, with an accumulation of cells in S-phase. The pharmacological effects of CPEC on freshly excised glioblastoma cells are quantitatively similar to those seen in a range of established tissue culture lines, including human glioma, colon carcinoma, and MOLT-4 lymphoblasts, supporting the recommendation that the drug may be advantageous for the treatment of human glioblastoma.