Identification of differentially expressed genes in breast cancer

A number of genes differentially expressed in breast cancer were isolated using a subtractive cloning technique. DNA sequence analysis and GenBank searches of T4F10, T2H7, and T2E5 cDNA clones found them to be identical with E2A, MSS1, and SEC13R genes. Their expression in a variety of primary breast tumor and cancer cell lines was compared with c-ERB-B2 and pS2 by Northern blot analysis. In breast cancer cell lines, the genes that we isolated are more frequently expressed than the previously described c-ERB-B2 and pS2.     

Evidence for an inherent,ATP-stimulated uptake of calcium into chromaffin granules

Summary Partially purified chromaffin granules (granular fraction), crude mitochondria (mitochondrial fraction) and a microsomal fraction were prepared from bovine adrenals by differential centrifugation and characterized by their catecholamine content, succinate dehydrogenase and glucose-6-phosphatase activity. During isotonic incubation with 0.1 mM ⁴⁵Ca²⁺ all fractions showed an uptake of ⁴⁵Ca²⁺, which was stimulated by ATP. In addition, after incubation the granular fraction was further fractionated by sucrose density gradient centrifugation (2.0–1.3 M sucrose; 170,000g·60 min). In some of these experiments the granular fraction was incubated simultaneously with ⁴⁵Ca²⁺ and [³H](-)noradrenaline to compare the uptake of both. The rate of uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction (characterized by the highest catecholamine content and the lowest succinate dehydrogenase activity) was doubled by ATP. The ATP-stimulated uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules was inhibited by N-ethylmaleimide (NEM) (0.1 mM), 2,4-dinitrophenol (DNP) (0.1 mM), azide (1 mM), carbonyl cyanide mchlorophenylhydrazone (CCCP) (20M), atractyloside (50 M), ruthenium red (40 M) and amobarbital (1 mM). This inhibition pattern was different from that of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into the microsomal fraction, but it was similar to that of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction. However, the following differences are incompatible with the view that a mitochondrial contamination, with a highly active uptake, is responsible for the ATP-stimulated uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules: a) The uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction was insensitive to 1 mM amobarbital, whereas this agent inhibited the uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules. b) Replacement of ATP by succinate stimulated the uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction only. c) The dependence of the ATP-stimulated uptake of ⁴⁵Ca²⁺ on the concentration of ATP (0.1–5 mM) was determined: while uptake into the 2.0 M sucrose fraction of chromaffin granules exhibited saturation kinetics, that into the mitochondrial fraction was linearly related to the concentration of ATP. Interestingly, uptake of ⁴⁵Ca²⁺ into those fractions of chromaffin granules that are known to be contaminated with mitochondria (1.6 M sucrose fraction) exhibited bot a saturable and a nonsaturable component. d) The uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction was more sensitive to 0.1 mM DNP than the uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules.The comparison of the ATP-stimulated uptake of ⁴⁵Ca²⁺ with that of [³H](-)noradrenaline into the 2.0 M sucrose fraction of chromaffin granules revealed that amobarbital, N,N-dicyclohexylcarbodiimide (DCCD), DNP and increasing concentrations of ATP had the same influence on both uptake processes. From our results we conclude that an inherent, ATP-stimulated uptake of ⁴⁵Ca²⁺ exists in chromaffin granules. The effects of agents on the uptake of ⁴⁵Ca²⁺ and/or [³H](-)noradrenaline into chromaffin granules are discussed with regard to the granular uptake mechanisms.This work was supported by the Deutsche ForschungsgemeinschaftSome results were reported at the 18th Spring Meeting 1977 of the Deutsche Pharmakologische Gesellschaft (Burger and Häusler 1977)     

Antiangiogenic properties of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin: an orally bioavailable heat shock protein 90 modulator.

PURPOSE: The purpose of this study was to investigate the antiangiogenic properties of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG; NSC707545), a water-soluble benzoquinone ansamycin. EXPERIMENTAL DESIGN: The activity of 17-DMAG, in vivo, was evaluated for inhibition of fibroblast growth factor (FGF)-2-induced angiogenesis in s.c. implanted Matrigel in mice. In vitro, the activity of 17-DMAG on endothelial cells (human umbilical vein endothelial cells; HUVEC) was tested in FGF-2; and vascular endothelial growth factor (VEGF)-induced proliferation and apoptosis, motility, and extracellular matrix invasion; and on the alignment of capillary like structures in Matrigel. The protein level of heat shock protein (Hsp)90 and client proteins was examined by Western blot in FGF-2 and VEGF-stimulated HUVEC. RESULTS: Daily oral administration of 17-DMAG affected the angiogenic response in Matrigel in a dose-dependent manner. The hemoglobin content in the Matrigel implants was significantly inhibited, and the histological analysis confirmed a decrease of CD31(+) endothelial cells and of structures organized in cord and erythrocyte-containing vessels. In vitro, the compound inhibited dose-dependently the migration and the extracellular matrix-invasiveness of HUVEC and their capacity to form capillary like structures in Matrigel. 17-DMAG treatment also inhibited FGF-2 and VEGF-induced HUVEC proliferation and resulted in apoptosis. Accordingly, the expression of Hsp90 direct client proteins (pAkt and c-Raf-1) or their downstream substrates including pERK was also affected. 17-DMAG consistently increased the expression of Hsp70. Throughout the study similar results were obtained with 17-allylamino-17-demethoxygeldanamycin (17-AAG; NSC330507), the analog compound currently undergoing clinical trials. CONCLUSIONS: We show that the Hsp90 targeting agents 17-DMAG and 17-AAG inhibit angiogenesis. The strong effects on endothelial cell functions, in vitro, indicate that the antiangiogenic activity of 17-DMAG/17-AAG could also be due to a direct effect on endothelial cells. The oral bioavailability of 17-DMAG might be of advantage in investigating the potential of this compound in clinical trials with antiangiogenic as well as antiproliferative endpoints.     

Pituicytoma: a distinctive low-grade glioma of the neurohypophysis

Pituicytoma is a rare, poorly characterized tumor of the sella and suprasellar region that is distinct morphologically from other local tumors and is thought to be derived from neurohypophyseal pituicytes. Clinical data, neuroimaging studies, and microsections were reviewed from nine such low-grade gliomas. Immunostains for glial, neuronal, and proliferation markers were performed on all nine tumors and six control neurohypophyses. Three tumors were studied ultrastructurally. Six men and three women, age 30 to 83 years (mean, 48 years), presented with visual symptoms, headache, or hypopituitarism. Magnetic resonance images showed solid, discrete, contrast-enhancing masses, four within the sella and five in the suprasellar space. The tumors consisted of sheets and/or fascicles of plump spindle cells with slightly fibrillar cytoplasm and slightly pleomorphic, oval-to-elongate nuclei with pinpoint nucleoli. Extracellular mucin was prominent in one tumor. Rosenthal fibers, granular bodies, and Herring bodies (granular axonal dilatations characteristic of the normal neurohypophysis) were lacking. Mitoses were rare or absent. MIB-1 labeling indices were low (0.5-2%). Tumor cells were strongly reactive for vimentin and S-100 protein, variably positive for glial fibrillary acidic protein, and nonreactive for synaptophysin and neurofilament protein. Cytoplasm varied in electron density and contained intermediate filaments. Neither meningothelial nor ependymal features were noted. Two tumors recurred at 20 and 26 months after subtotal resection, but none of the six completely resected tumors have done so. Pituicytomas are discrete, largely noninfiltrative low-grade gliomas of the sellar region that occur in adults. Their histologic appearance is distinct from pilocytic and ordinary, infiltrative astrocytomas. The distinction between pituicytoma and normal neurohypophysis is aided by the latter's content of axons, Herring bodies, and perivascular anucleate zones rich in axonal terminations. Although curable by total excision, subtotal resection can be associated with recurrence.     

Comparing sulindac with indomethacin for closure of ductus arteriosus in preterm infants

Objectives: A prospective study comparing the efficiacy and side-effects of oral sulindac with intravenous indomethacin in clinically stable preterm infants (<1750 g) requiring non-invasive closure of haemodynamically significant patent ductus arteriosus.
Methodology: As maturity and birthweight are the two major determinants of ductal closure, infants were matched as closely as possible for these parameters. An eligible patient was first assigned to the sulindac group and a subsequent patient with similar gestational age (± 1 week) and birthweight (±100 g) to the previously recruited infant would automatically receive indomethacin. A total of eight infants were enrolled in each group.
Results: The ductus arteriosus was successfully closed in all eight infants receiving indomethacin, and in seven of eight infants receiving sulindac. No significant differences were found with regards to the ductal size between the two groups at diagnosis or on each of the consecutive days of treatment ( P >0.25). More renal adverse effects were encountered in the indomethacin group. Significant differences in changes from baseline value for urine output, plasma sodium, urea and creatinine concentrations were noted at 24, 48 and 72 h after commencement of treatment between the two groups ( P <0.05). All the parameters returned to normal or pre-treatment levels 48 h after stopping therapy. Unexpectedly, severe gastrointestinal complications were encountered in the sulindac group.
Conclusions: Sulindac is capable of promoting ductal constriction in clinically stable preterm infants without compromising the renal function. The spectrum of gastrointestinal complications observed in sulindac treated infants were similar to those described for indomethacin. The use of sulindac for ductal closure in the preterm infant should remain experimental.     

Aberrant crypt focus promotion and glucose intolerance: correlation in the rat across diets differing in fat, n-3 fatty acids and energy

McKeown-Eyssen (Cancer Epidemiol. Biomarkers Prevent., 3, 687-695, 1994) and Giovannucci (Cancer Causes Control, 6, 164-179, 1995), noting the striking similarity in lifestyle risk factors for colorectal cancer and insulin resistance, proposed that the hyperinsulinemia, glycemia and hypertriglyceridemia associated with insulin resistance promotes colon cancer. To compare the effect of diet on colon cancer promotion and insulin resistance in the F344 rat, we assessed the effect of fat, n-3 fatty acids and energy in pairwise comparisons on average size of aberrant crypt foci (ACF) and on glucose intolerance in the same animals in a single experiment. Diets high in fat and energy increased and diets with increased n-3 fatty acids and calorie restriction decreased both ACF growth and glucose intolerance compared with control diets. The measures of promotion of colon cancer and insulin resistance were strongly correlated (n = 98, r = 0.67, P < 0.001). In addition, both were highly correlated with daily energy intake (r = 0.62 and 0.66) and were also correlated with basal (post-prandial) insulin, glucose and triglycerides (r = 0.31-0.53, P < 0.01). We concluded that ACF growth and glucose intolerance are correlated for a wide range of diets and that increased circulating energy (glucose and triglycerides) may lead to both colon cancer promotion and insulin resistance.      

Expression of p53, Bcl-2 and Bax in cisplatin-induced apoptosis in testicular germ cell tumour cell lines.

We examined the sensitivity for cisplatin-induced apoptosis in a panel of four testicular germ cell tumour (TGCT) cell lines and monitored the cellular expression of the apoptosis-related proteins p53, Bcl-2 and Bax. Three of four TGCT cell lines (NT2, NCCIT and S2) were hypersensitive for cisplatin-induced apoptosis, while the TGCT cell line 2102 EP appeared to be resistant for cisplatin-induced apoptosis, even at relatively high drug concentrations (12.5 microM). For all four cell lines, the induction of apoptosis by cisplatin correlated with drug sensitivity in the MTT assay. The differences in chemosensitivity and induction of apoptosis could not be attributed to differences in cellular platinum accumulation, DNA platination or platinum-DNA adduct removal. We next analysed the relationship between p53 status and cisplatin-induced up-regulation of p53, and the susceptibility to cisplatin-induced apoptosis. Wild-type p53 containing NT2 and 2102 EP cells showed p53 up-regulation upon drug treatment, and NCCIT (mutant p53) and S2 (no p53 protein) cells did not. Consistently, the increase in wild-type p53 protein in NT2 and 2102 EP cells led to an increase in mRNA level of the p53 downstream gene p21/WAF/CIP, whereas mutant p53-containing NCCIT cells and p53-non-expressing S2 cells could not transactivate this p53-responsive gene. As NT2, NCCIT and S2 were readily triggered into apoptosis, while 2102 EP cells failed to undergo cisplatin-induced apoptosis, our data suggest that the presence of wild-type and/or transactivation-competent p53 might not be an absolute prerequisite for efficient induction of apoptosis in TGCT cell lines. Also endogenous levels of Bcl-2 and Bax expression did not correlate with cisplatin-induced apoptosis. In addition, the endogenous Bcl-2 and Bax expression was not affected by cisplatin treatment. The present study suggests that, at least in our panel of TGCT cell lines, hypersensitivity for cisplatin-induced apoptosis might not be necessarily correlated with the presence of wild-type p53 and is probably not associated with Bcl-2 and Bax expression.     

Angiogenesis is an early event in the development of chemically induced skin tumors

In this study we have analyzed the vascular response induced in the two- stage carcinogenesis model in SENCAR mice. The role of angiogenesis has not been explored in this model, which is the paradigm of multistage carcinogenesis and a model for neoplastic lesions derived from exophytic premalignant lesions (e.g. colon carcinoma, bladder papilloma). We investigated if angiogenesis is involved in the formation of papillomas and in the progression from papilloma to carcinoma. To this end we analyzed the vasculature of normal and hyperplastic skin, focal epidermal hyperplasias that are precursors of papillomas, papillomas at different stages and squamous cell carcinomas. We also analyzed the vascularization of papillomas induced in two strains of mice that differ in their susceptibility to malignant progression. We show here that angiogenesis is turned on in the earliest stages of papilloma formation. In late stages, regardless of state of progression, the predominant response is an increase in the size of blood vessels. Thus, in the SENCAR mouse model, representative of exophytic tumors, the angiogenesis switch is a very early event, probably mechanistically related to the development of the primarily exophytic lesions. Therefore, the density of blood vessels cannot be used as a predictor of malignant progression in this model.      

Simplified quantitative determination of cerebral perfusion reserve with H2(15)O PET and acetazolamide

The measurement of regional cerebral blood flow (rCBF) and perfusion reserve (PR) with H2(15)O positron emission tomography (PET) and acetazolamide challenge is of importance in evaluating patients with cerebrovascular disease and is thought to be useful in selecting patients for possible vascular surgery. Full quantitative assessment of rCBF with PET requires arterial blood sampling, which is inconvenient in a clinical setting. In this work, we present a simple non-invasive method with which to quantitatively evaluate PR in one PET session lasting no more than 30 min. In ten patients with cerebrovascular disease, rCBF was measured with H2(15)O PET under the baseline condition and after administration of 1 g acetazolamide using a standard technique involving arterial blood sampling. The activity accumulated over 60 s was normalized to injected activity per kilogram body weight (nAA) and compared with rCBF in eight different brain regions. A high linear correlation was found for PR based on nAA (PRnAA) and rCBF (PRrCBF) (PRnAA=0.843 PRrCBF + 0.092, r=-0.83, Pearson's correlation coefficient). Bland-Altman analyses further confirmed that PRnAA reflects PR in a quantitative manner. These results demonstrate that the method based on normalized counts allows the quantitative assessment of PR without blood sampling.