Integrated genomic analyses identify WEE1 as a critical mediator of cell fate and a novel therapeutic target in acute myeloid leukemia.

Acute myeloid leukemia (AML) remains a therapeutic challenge despite increasing knowledge about the molecular origins of the disease, as the mechanisms of AML cell escape from chemotherapy remain poorly defined. We hypothesized that AML cells are addicted to molecular pathways in the context of chemotherapy and used complementary approaches to identify these addictions. Using novel molecular and computational approaches, we performed genome-wide short-hairpin RNA screens to identify proteins that mediate AML cell fate after cytarabine exposure; gene expression profiling of AML cells exposed to cytarabine to identify genes with induced expression in this context; and examination of existing gene expression data from primary patient samples. Integration of these independent analyses strongly implicates cell-cycle checkpoint proteins, particularly WEE1, as critical mediators of AML cell survival after cytarabine exposure. Knockdown of WEE1 in a secondary screen confirmed its role in AML cell survival. Pharmacologic inhibition of WEE1 in AML cell lines and primary cells is synergistic with cytarabine. Further experiments demonstrate that inhibition of WEE1 prevents S-phase arrest induced by cytarabine, broadening the functions of WEE1 that may be exploited therapeutically. These data highlight the power of integrating functional and descriptive genomics, and identify WEE1 as a potential therapeutic target in AML.

Alpha 1B-adrenergic receptors in rat renal microvessels.

Although several alpha-adrenergic receptor genes are expressed in the rat kidney, their expression in the renal vasculature has not been studied. Since pharmacological studies have suggested that an alpha 1B-adrenergic receptor may mediate renal vasoconstriction, we studied the expression of alpha 1B-adrenergic receptors in renal microvessels, from 10- to 14-week-old male spontaneously hypertensive rats (SHR) and their normotensive control, the Wistar-Kyoto rat (WKY). In these microvessels, isolated by perfusion with iron, alpha 1B-adrenergic receptor mRNA levels (by ribonuclease protection assay) were similar in SHR and WKY rats. Photo-affinity labeling with [125I]-arylazidoprazosin demonstrated the presence of alpha 1B-adrenergic receptor protein. Maximum receptor density (determined by 3H-prazosin binding: Bmax 59.8 +/- 4.1 and 58.7 +/- 4.3; Kd 0.48 +/- 0.05 nM and 0.31 +/- 0.06 nM in SHR and WKY, respectively) and chloroethylclonidine (CEC)-sensitive binding sites (determined by [125I]-(2-beta(4-hydroxyphenyl)-ethylaminomethyl)-tetralone binding) (125I-HEAT) were similar in SHR and WKY rats. There are two novel findings in these studies: (1) the alpha 1B-adrenergic receptor gene is expressed in renal microvessels of WKY and SHR; (2) alpha 1B-adrenergic receptor gene expression in renal microvessels is not altered in adult SHR. The failure to down-regulate expression of the alpha 1B-adrenergic receptor at the mRNA and protein level in the SHR could result in persistence of alpha 1B-adrenergic receptor effects and contribute to the increased vascular resistance in hypertension.

Ontogenic changes and regulation of renal angiotensin II type 1 receptor gene expression during fetal and newborn life.

Factors regulating the expression of the angiotensin II subtype 1 (AT1) receptor during fetal life have not been investigated previously. The present study was designed 1) to characterize the ontogeny of AT1 receptor gene expression in the kidney of fetal and newborn sheep and 2) to determine the influence of both glucocorticoids and renal nerves in modulating AT1 gene expression during fetal life and during the transition from fetal to newborn life. We first isolated and cloned a PCR product that has 98 and 94% homology with the cDNA encoding the bovine and pig AT1 receptors, respectively, and 99 and 98% homology with the corresponding deduced protein sequences. Probing with this cDNA, we demonstrated that renal AT1 mRNA expression did not change significantly during the last trimester of gestation in fetal sheep or immediately after birth but decreased significantly 10 d after birth. We also demonstrated that renal denervation in the fetus had no effect on renal AT1 gene expression in 24-h-old newborn lambs. On the other hand, we observed in 130-d twin fetuses that a continuous intraperitoneal infusion (1 mL/h) of cortisol (3 mg/h or 6.2 mumol/h) for 48 h in one of the twins increased the fetal plasma cortisol concentration from 32.0 +/- 7.1 to 1126 +/- 231 nmol/L and produced a significant decrease (p < 0.005) in renal AT1 gene expression compared with the control twin receiving an intraperitoneal infusion of 0.9% NaCl. In summary, this study demonstrates that renal AT1 gene expression is elevated during fetal life and decreases after birth. It is also shown that glucocorticoids, but not renal nerves, contribute to the regulation of renal AT1 gene expression during development.

Developmental regulation of the alpha 1B-adrenoceptor in the sheep kidney.

The expression of renal alpha 1B-adrenoceptor (alpha 1B-AR) mRNA was studied and contrasted with the expression of renal renin mRNA in fetal and newborn sheep. Fetal sheep between 90 and 91, 116 and 118, and 139 and 141 d gestation (term is 145 d gestation) as well as newborn lambs between 1 and 2 d old and 8 and 10 d old were studied (n = 3 for each age range). The role of the renal nerves in regulating changes in alpha 1B-AR gene expression was also investigated by measuring renal cortical alpha 1B-AR mRNA levels and receptor kd and maximum number of binding sites in 24-h-old lambs that were either denervated (n = 6) or sham-operated (n = 5) 3 d before birth. During development, renal alpha 1B-AR mRNA levels show a marked increase in term fetuses; this increase persists into the first 2 d of life and is distinct from the developmental pattern seen for renal renin mRNA levels. Denervation of term fetuses does not alter the expression of renal alpha 1B-AR mRNA in newborn lambs when compared with sham-operated controls but decreases significantly the expression of the renin gene (p < 0.05). These results suggest that the alpha 1B-AR gene is developmentally regulated in the kidney in a pattern distinct from that seen for renin.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemolytic-uremic syndrome: a population-based study in Washington, DC and Baltimore, Maryland.

A population-based study of hemolytic-uremic syndrome (HUS) revealed that 20 child residents of Washington, DC and Baltimore, Maryland were hospitalized with HUS from January 1979 through September 1983. The number of cases peaked during the summer and fall; none occurred during the winter. Incidence of hospitalized cases was higher in Whites and girls than in Blacks or boys, and the average annual incidence was 1.08 cases/100,000 children less than 5 year old. This study demonstrates that HUS is not unique to the West Coast, as previously suggested.

Pancreatic endocrine insufficiency in posttransplant cystinosis.

Nephropathic cystinosis causes renal death by approximately age 10 years. With increased life span due to kidney transplantation, ten to 25 years of cystine accumulation has resulted in pancreatic complications in individuals with cystinosis. We noted severe hyperglycemia in five posttransplant patients, three of whom remained insulin-dependent diabetics several years after transplant. The clinical findings were not consistent with steroid-dependent or insulin-resistant diabetes. Pancreatic cystine deposition was detected histologically and biochemically on post-mortem examination of two other patients. We conclude that hyperglycemia may be anticipated in the immediate posttransplant period in cystinotic patients and that some patients will require insulin therapy years later. The use of cystine-depleting agents should be considered in posttransplant cystinosis as an attempt to prevent potential damage to the pancreas and other organs from cystine deposition.

Accelerated recovery from immune-mediated thrombocytopenia with plasmapheresis.

Autoimmune thrombocytopenia unresponsive to corticosteroid therapy developed in a 16-year-old female with long-standing Sjögren's syndrome. Serial plasma exchange caused a linear decrease in platelet antibody titer associated with a concomitant rise in platelet count. Statistical analysis of sequential platelet counts revealed an increase with plasmapheresis and immunosuppression that was significantly greater than that achieved with immunosuppression alone (p less than 0.005).

6-Chloro-2(1-piperazinyl) quinoxaline (CPQ): action on serotonin-induced behavioral responses and interactions with chloromethamphetamine and dimethyl-meta-tyramine.

6-Chloro-2(1-piperazinyl) quinoxaline (CPQ) was examined pharmacologically and biochemically as an inhibitor of the neuronal reuptake of serotonin, dopamine and norepinephrine. The compound was 25-50 times more potent than chlorimipramine in potentiating the head-twitch response to 5-hydroxytryptophan (5-HTP) and in antagonizing p-chloromethamphetamine (PCMA)-induced depletion of brain serotonin in rats. CPQ also potentiated the forepaw clonus produced by 5HTP in rats and antagonized PCMA-induced head twitches. At dose levels 30 times those necessary to significantly affect serotoninergic systems, CPQ was ineffective in antagonizing either tetrabenazine-induced sedation in mice or the depletion of rat brain and heart norepinephrine or brain dopamine produced by 4, alpha-dimethylmeta-tyramine (H77/77). The data indicate that CPQ exhibits a high degree of potency and selectivity in inhibiting the neuronal reuptake of serotonin.

The effect of alcohol upon "muscle pull" of mice.

Equipment is described for measuring the pull exerted by mice to escape tail restraint and enter a black box. Alcohol, at doses exceeding about 2.5 g/kg, intraperitoneally, significantly decreased "muscle pull", measured 5-60 min after administration. Fifty per cent depression of muscle pull was obtained with doses of about 3.04, 3.18 and 3.55 g/kg of alcohol, 15, 30 and 60 min after alcohol administration, respectively. Depression of muscle pull correlated with alcohol concentrations in the blood plasma. The data show that muscle pull was decreased in only a few animals with plasma alcohol concentrations of less than about 250 mg%, but was significantly depressed in animals with plasma alcohol concentrations of 350 mg% or more. Although differing from it in some respects, the method described is similar to the tilting-plane method which has been used in studies dealing with the effect of alcohol in rats and mice.

Human metabolism of cyproheptadine.

Gas-liquid chromatographic and mass, nuclear magnetic resonance, and infrared spectrometric techniques were utilized to identify some of the metabolites of cyproheptadine in the urine of human subjects who had ingested radiolabeled drug. Aromatic ring hydroxylation (followed by glucuronide conjugation), N-demethylation, and heterocyclic ring oxidation were shown to occur in man. The principal metabolite, however, was identified tentatively as a quaternary ammonium, glucuronide-like conjugate of cyproheptadine. No evidence was found for metabolic changes at the tricyclic ethylene bridge in this species.