The mechanism of action of many chemotherapeutic agents targets the cell cycle. Recently, we demonstrated cytotoxic and other anti-tumor effects of flavopiridol, the first synthetic cyclin dependent kinase (CDK) inhibitor to enter clinical trials, on the murine GL261 glioma cell line in vitro (Newcomb et al., Cell Cycle 2003; 2:243). Given that flavopiridol has demonstrated anti-tumor activity in several human xenograft models, we wanted to evaluate it for anti-glioma activity in vivo in our established subcutaneous and intracranial GL261 experimental tumor models. In particular, the intracranial animal model recapitulates many of the histopathological and biological features of human high-grade glioma including both necrosis with pseudopalisading and invasion of the brain adjacent to tumor. Here we tested the activity of flavopiridol against tumors formed by GL261 cells, first as subcutaneous implants, and then in the intracranial model. We demonstrate efficacy of flavopiridol as a single modality treatment in delaying tumor growth in both animal models. We hypothesize that flavopiridol treatment induced tumor growth delay by two possible mechanisms involving growth arrest combined with recruitment of tumor cells to S-phase. Based on our findings, flavopiridol should be considered as a treatment approach for patients with high-grade glioma. © L a n d e s B i o s c i e n c e 2 0 0 4. N o t f o r d i s t r i b u t i o n .

ABSTRACfFenoldopam mesylate (PM), a selective post-junctional dopaminergic (DA,) vasodilator, causes lesions of large caliber splanchnic arteries in the rat characterized by necrosis of medial smooth muscle cells and hemorrhage. FM does not induce lesions in other vascular beds of the rat, or in dogs or monkeys. Dopamine, like FM, causes hemorrhagic lesions of large caliber splanchnic arteries in the rat, as well as fibrinoid necrosis of small caliber arteries (< 100 Jlm) of the splanchnic, cerebral, coronary and renal vascular beds. Dopamine is an alpha-and beta-adrenoceptor and a dopaminergic receptor agonist. Because these arterial lesions are thought to result from the pharmacologic activity of these 2 compounds; we sought to ascertain the presence of DA, receptors in mesenteric arteries of the rat and to determine the role of these or other vascular receptor subtypes in lesion induction. We also studied the process of repair after arterial injury caused by FM or dopamine. The presence of DA 1 receptors was confirmed in isolated perfused mesenteric arteries by standard pharmacologic techniques; stimulation by PM resulted in vasodilation which was inhibited by the DA) receptor antagonist SK&F 83566-C. Likewise, SK&F 83566-C prevented the induction of hemorrhagic lesions of large caliber arteries in rats upon infusion of PM or dopamine. In rats co-exposed to the alpha-adrenoreceptor antagonist phenoxybenzamine (PBZ) and either FM or dopamine, the incidence and severity of hemorrhagic lesions oflarge caliber arteries were increased, but PBZ prevented the formation of dopamine-induced fibrinoid lesions in arteries ofsmall caliber. Rats exposed concurrently to dopamine, phenoxybenzamine, and SK&F 83566-C were free of all arterial lesions. Thus, the induction of splanchnic arterial lesions in the rat by dopamine and PM is caused by stimulation of, and interaction between, alpha-adrenoceptors and dopaminergic DA 1 receptors. Fibrinoid lesions of small arteries (alphaadrenoceptor-mediated) were repaired, as observed morphologically by 14 d after exposure to dopamine. Hemorrhagic lesions of large caliber arteries (DA) receptor-mediated) had undergone significant repair by 28 d after exposure to PM but these arteries possessed a thicker media surrounded by adventitial fibrosis. Thus, morphologically distinct receptor-mediated splanchnic arterial lesions induced by dopaminergic and alpha-adrenoceptor agonists follow a markedly different course of repair. Arterial lesions induced by FM or dopamine by activation ofpost-junctional dopaminergic DA, receptors may represent a model of polyarteritis nodosa.

To regulate ionic and fluid homeostasis, the colon relies upon a series of Na(+)-dependent transport proteins. Recent studies have identified a sodium/hydrogen exchanger (NHE) 4 (NHE4) protein in the gastrointestinal tract but to date there has been little description of its function. Additionally, we have previously shown that aldosterone can rapidly modulate Na(+)-dependent proton excretion via NHE proteins. In this study we examined the role of NHE4 in rat and human colonic crypts, determined the effect of aldosterone on NHE4 specifically, and explored the intracellular pathways leading to activation. Colonic samples were dissected from Sprague-Dawley rats. Human specimens were obtained from patients undergoing elective colon resections. Crypts were isolated using ethylenediaminetetraacetic acid and intracellular pH (pH(i)) changes were monitored using 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Crypts were exposed to 7 μM ethylisopropylamiloride or 400 μM amiloride, doses previously shown to inhibit NHE1 and NHE3 but allow NHE4 to remain active. Functional NHE4 activity was demonstrated in both rat and human colonic crypts. NHE4 activity was increased in the presence of 1 μM aldosterone. In the rat model, crypts were exposed to 100 μM 3-isobutyl-1-methylxanthine/1 μM forskolin and demonstrated a decrease in NHE4 activity with increased cAMP levels. No significant change in NHE4 activity was seen by increasing osmolarity. These results demonstrate functional NHE4 activity in the rat and human colon and an increase in activity by aldosterone. This novel exchanger is capable of modulating intracellular pH over a wide pH spectrum and may play an important role in maintaining cellular pH homeostasis.

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General ProcedureThe studies reported herein were, with one exception, all done in the same laboratory. They were intended to delineate a toxicologic profile for these three goldcontaining compounds, on which assessment of their safety for human clinical trials would be based.Young Charles River (CD) male and female rats were used. They were randomly divided into the appropriate dose groups, maintained in wire-mesh cages, 2 per unit except the last odd-numbered rat in each group, in an air-conditioned room at 72 f 2'F temperature and 50% f 5% relative humidity. Tap water and Purina Laboratory Chow@ were available at all times.The rats were weighed before each dosing to determine the correct dose-volume to be administered. Observations of general health and clinical signs of toxicity were made daily. Body weight and 24 hour food consumption were recorded weekly in the short-term studies and somewhat less frequently toward the end of the longer studies.Ophthalmoscopic examinations were done on all rats before commencement of drug administration and at the end of the study. In the 12-month studies examinations were also done at the 6 month interval.Clinicopathologic examinations (hematologic, clinical chemical and uroanalytic) were done once or twice before commencement of dosing, at the I-week, I-, 3-, 6-, 9-, and 12-month intervals (depending on the length of the study) and at the end of the study.Hematologic examinations included hemoglobin, packed-cell volume and total and differential leukocyte counts and, in some studies, the red blood cell count and red-cell indices.The clinical biochemical determinations included the plasma glucose, urea nitrogen, alkaline phosphatase and glutamic-pyruvic transaminase tests. The uroanalytic determinations included pH and the presence of glucose, ketones, bilirubin and occult blood.At the end of each study, a postmortem examination was done on each surviving rat. A postmortem and histologic examination were done on all rats that died during a study except where precluded by autolysis. Sections were prepared from all gross lesions except certain very common lesions of natural disease. A histologic examination was done of the heart, aorta, lung, thyroid, parathyroid, adrenal, lymph node, ovary, pancreas, muscle, nerve, liver, spleen, stomach, intestines, urinary bladder, salivary gland, bone, bone marrow, skin, mammary gland, brain, cord and eye from 6 males and 6 females from the control and highest dose group of each test compound. When a treatment-related lesion occurred, the affected tissue