Androgen-ablation is a most commonly prescribed treatment for metastatic prostate cancer but it is not curative. Development of new strategies for treatment of prostate cancer is limited partly by a lack of full understanding of the mechanism by which androgen regulates prostate cancer cell proliferation. This is due, mainly, to the limitations in currently available experimental models to distinguish androgen/androgen receptor (AR)-induced events specific to proliferation from those that are required for cell viability. We have, therefore, developed an experimental model system in which both androgen-sensitive (LNCaP) and androgen-independent (DU145) prostate cancer cells can be reversibly blocked in G(0)/G(1) phase of cell cycle by isoleucine deprivation without affecting their viability. Pulse-labeling studies with (3)H-thymidine indicated that isoleucine-deprivation caused LNCaP and DU145 cells to arrest at a point in G(1) phase which is 12-15 and 6-8 h, respectively, before the start of S phase and that their progression into S phase was dependent on serum factors. Furthermore, LNCaP, but not DU145, cells required AR activity for progression from G(1) into S phase. Western blot analysis of the cell extracts prepared at regular intervals following release from isoleucine-block revealed remarkable differences in the expression of cyclin E, p21(Cip1), p27(Kip1), and Rb at the protein level between LNCaP and DU145 cells during progression from G(1) into S phase. However, in both cell types Cdk-2 activity associated with cyclin E and cyclin A showed an increase only when the cells transited from G(1) into S phase. These observations were further corroborated by studies using exponentially growing cells that were enriched in specific phases of the cell cycle by centrifugal elutriation. These studies demonstrate usefulness of the isoleucine-deprivation method for synchronization of androgen-sensitive and androgen-independent prostate cancer cells, and for examining the role of androgen and AR in progression of androgen-sensitive prostate cancer cells from G(1) into S phase.

Background and Purpose-Variations in blood-brain barrier (BBB) opening after ischemia have been suggested by some tracer and magnetization transfer studies, although direct in vivo proof is still lacking. Contrast-enhanced magnetic resonance imaging (MRI) is also often used to visualize BBB damage in stroke. We hypothesized that MR contrast agents of different sizes enhance differently when BBB openings vary in size and that magnetization transfer alterations, measured by T 1 in the presence of off-resonance radiofrequency saturation (T 1sat ), in these regions reflect such differences. Methods-Male Wistar rats (Ϸ300 g, nϭ7) were subjected to 3 hours of suture occlusion of the middle cerebral artery followed by reperfusion. Status of the BBB at 24 hours after the ictus was assessed first by Gd-DTPA (554 Da) MRI and then by Gd-bovine serum albumin linked to Evans blue (Gd-BSA-EB; Ϸ68 kDa) MRI for contrast enhancement; T 1sat changes, cerebral blood flow, and blood-to-brain transfer constants (K i s) for the 2 contrast agents were measured. After MRI, rats were injected with fluorescent dextran and brains were studied by fluorescence microscopy. Results-The Gd-BSA-EB-enhancing areas were always smaller (147Ϯ80 pixels) than those for Gd-DTPA (308Ϯ204 pixels) and were contained within the latter. The difference between the 2 areas was significant (Pϭ0.024). Changes in T 1sat were larger in Gd-BSA-EB-enhancing areas (ipsilateral to contralateral [I/C]ϭ1.53Ϯ0.20) than in Gd-DTPAenhancing areas (I/Cϭ1.40Ϯ0.24, Pϭ0.005). The differences in cerebral blood flow values between the 2 regions were not significant (Pϭ0.62), but those for the K i values of the 2 tracers were different (Pϭ0.01 to 0.02). Excellent agreement between regions of Gd-BSA-EB enhancement and EB fluorescence was also observed. Conclusions-These results substantiate earlier reports of regional differences in BBB opening after stroke and provide the first in vivo evidence for this phenomenon. They also support the possible use of T 1sat in quantifying stroke-induced graded BBB damage in the absence of contrast-enhanced MRI. (Stroke. 2008;39:427-432.)

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Human osteosarcoma and mammary carcinoma cells were cultured separately in a medium supplemented with fetal calf serum, until they were confluent. The medium was then replaced by serum-free medium supplemented with heparin. Both cell cultures secreted collagenase, and this activity was inhibited by a cartilage-derived protein of low molecular weight. Since cartilage is rarely invaded by neoplasms, the presence of this inhibitor may play an important role in the regulation of tumor invasion.