We have investigated the regulation of p27 kip1 , a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence (G 0 ) to a proliferative (G 1 ) state. The level of p27 kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27 kip1, as well as a transient increase in cyclin D1-associated p27 kip1 that later declines concomitantly with the loss of total p27 kip1 . Analysis of metabolically labelled cells revealed that cyclin D2, cyclin D3, and cdk4 were also partnered with p27 kip1 in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growth factor (PDGF) treatment. Furthermore, the decline in p27 kip1 and reduced association with cyclin D3, initiated by the addition of PDGF but not plasma-derived factors, suggested that these changes are involved in competence, the first step in the exit from G 0 . Synthesis of p27 kip1 as determined by incorporation of [ 35 S]methionine was repressed upon mitogenic stimulation, and PDGF was sufficient to elicit this repression within 2 to 3 h. Pulse-chase experiments demonstrated the reduced rate of synthesis was not the result of an increased rate of degradation. Full repression of p27 kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor 5,6-dichlorobenzimidazole riboside. These characteristics demonstrate that repression was a late effect of PDGF and was consistent with our finding that conditional expression of activated H-ras did not affect synthesis of p27 kip1. Northern (RNA) analysis of p27 kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27 kip1 mRNA, suggesting that the PDGF-regulated decrease in p27 kip1 expression occurred through a translational mechanism.Under normal circumstances, proliferation of mammalian cells is a highly controlled process. This control is achieved, in part, by growth factors and cytokines that exert either mitogenic and/or antiproliferative effects in a cell-specific manner (1). Binding of growth-regulatory ligands results in the activation of signaling pathways that trigger direct alteration of specific metabolic pathways as well as immediate and delayed changes in gene expression (65). Mitogenic stimulation initiates a program of sequential synthesis of proteins, termed cyclins, that complex with and activate cyclin-dependent kinases (cdks) (9, 29-31, 39, 52, 54, 64, 67), enzymes that modulate key regulatory events leading to progression through and transitions between different stages of the cell cycle (7,13,20,21,47,55,64). The order of cyclin synthesis during cell cycle traverse has been examined in a variety of cells, including T cells, macrophages, epithelial cells, and fibroblasts (16,30,31,44,45,50,52). The D-type cyclins, which complex with cdk4 and cdk6, are the first cyclins synthesized during the cell cycle. They are detected in mid-G 1 and are believed to function as Rb ...

Findings strongly suggest abnormal differentiation in the interstitial cystitis urothelium with a loss of barrier function markers and altered differentiation markers being independent and occurring independently of inflammation. Loss of the glycosaminoglycan layer was associated with a loss of biglycan and perlecan on the luminal layer.

Most cancer patients die with metastatic disease, thus, good models that recapitulate the natural process of metastasis including a dormancy period with micrometastatic cells would be beneficial in developing treatment strategies. Herein we report a model of natural metastasis that balances time to complete experiments with a reasonable dormancy period, which can be used to better study metastatic progression. The basis for the model is a 4T1 triple negative syngeneic breast cancer model without resection of the primary tumor. A cell titration from 500 to 15,000 GFP tagged 4T1 cells implanted into fat pad number four of immune proficient eight week female BALB/cJ mice optimized speed of the model while possessing metastatic processes including dormancy and beginning of reactivation. The frequency of primary tumors was less than 50% in animals implanted with 500–1500 cells. Although implantation with over 10,000 cells resulted in 100% primary tumor development, the tumors and macrometastases formed were highly aggressive, lacked dormancy, and offered no opportunity for treatment. Implantation of 7,500 cells resulted in >90% tumor take by 10 days; in 30–60 micrometastases in the lung (with many animals also having 2–30 brain micrometastases) two weeks post-implantation, with the first small macrometastases present at five weeks; many animals displaying macrometastases at five weeks and animals becoming moribund by six weeks post-implantation. Using the optimum of 7,500 cells the efficacy of a chemotherapeutic agent for breast cancer, doxorubicin, given at its maximal tolerated dose (MTD; 1 mg/kg weekly) was tested for an effect on metastasis. Doxorubicin treatment significantly reduced primary tumor growth and lung micrometastases but the number of macrometastases at experiment end was not significantly affected. This model should prove useful for development of drugs to target metastasis and to study the biology of metastasis.

Interstitial cystitis (IC) is a chronic and painful bladder syndrome of unknown cause with no reliable biological marker or effective therapy. Vascular endothelial growth factor (VEGF), which plays a key role in bladder inflammation, is closely associated with the vascular alterations observed in patients with IC. However, our recent findings of VEGF receptors (VEGF-Rs) and VEGF coreceptors on nonendothelial cells in human and mouse urothelium suggest that additional VEGF targets and functions are possible in IC bladders. We report here that VEGF-Rs and coreceptors (neuropilins; NRP) are strongly expressed in both the human bladder urothelium and in the human bladder cancer cell line (J82) and that the expression of NRP2 and VEGF-R1 is significantly downregulated in IC compared with control subjects. In addition, treatment of J82 cells with bacillus Calmette-Guérin (BCG), a novel treatment strategy for IC, upregulates the messages for NRPs and VEGF-Rs. Furthermore, intravesical instillation of an internalizable VEGF fluorescent tracer (scVEGF/Cy5.5) into mouse urinary bladders results in a marked ligand accumulation in the urothelium and bladder parenchyma, indicating that urothelial VEGF-Rs are functionally active and capable of ligand interaction and internalization. Our results suggest that the VEGF pathway is altered in IC, that urinary VEGF may gain access to the bladder wall via these receptors, and that BCG treatment may replenish the missing VEGF-Rs/NRP receptors. Together, these results suggest that levels of NRPs, VEGF-Rs, and VEGF are new putative markers for the diagnosis of IC and that modulating these receptors can be exploited as therapeutic strategies.

Purpose Chondroitin sulfate, which is less expensive and more inert than heparinoids, hyaluronan or pentosan polysulfate, has been introduced to restore the barrier function lost due to epithelial dysfunction in interstitial cystitis (IC). The binding of chondroitin sulfate to damaged bladder as a function of the range of pH seen in urine, its efficacy in restoring the bladder's permeability barrier, and the capacity of damaged bladder to bind chondroitin sulfate have not been determined previously. Methods Binding of chondroitin sulfate to bladder urothelium was investigated quantitatively using chondroitin sulfate highly labeled with Texas Red and quantitative fluorescence microscopy in a mouse model of acid damage of the urothelium. The efficacy of restoring the barrier function was determined using passage of intravesically instilled 86Rb, a potassium ion mimetic, through the urothelium into the bloodstream in a rat model of bladder damage. The binding capacity of acid-damaged bladder was determined by fluorometry. Results Chondroitin sulfate bound tightly and exclusively to the mouse bladder surface that had been damaged by acid but showed only minimal binding to undamaged bladder. There was no systematic variation with pH. The model showed some variability in the degree of damage induced. In rats, chondroitin sulfate instillation restored permeability to 86Rb to control levels. Binding was saturable at 0.67 ± 0.13 mg/cm2 of bladder surface. Conclusions Chondroitin sulfate binds preferentially to damaged urothelium and restores the impermeability barrier. This suggests that the GAG layer is a major contributor to the impermeability of bladder urothelium. As determined by the binding capacity, the dose applied to humans in Canada (400 mg per instillation) is sufficient to obtain maximum efficacy.

No abstract

A major problem in cancer research is the lack of a tractable model for delayed metastasis. Herein we show that cancer cells suppressed by SISgel, a gel-forming normal ECM material derived from Small Intestine Submucosa (SIS), in flank xenografts show properties of suppression and re-activation that are very similar to normal delayed metastasis and suggest these suppressed cells can serve as a novel model for developing therapeutics to target micrometastases or suppressed cancer cells. Co-injection with SISgel suppressed the malignant phenotype of highly invasive J82 bladder cancer cells and highly metastatic JB-V bladder cancer cells in nude mouse flank xenografts. Cells could remain viable up to 120 days without forming tumors and appeared much more highly differentiated and less atypical than tumors from cells co-injected with Matrigel. In 40% of SISgel xenografts, growth resumed in the malignant phenotype after a period of suppression or dormancy for at least 30 days and was more likely with implantation of 3 million or more cells. Ordinary Type I collagen did not suppress malignant growth, and tumors developed about as well with collagen as with Matrigel. A clear signal in gene expression over different cell lines was not seen by transcriptome microarray analysis, but in contrast, Reverse Phase Protein Analysis of 250 proteins across 4 cell lines identified Integrin Linked Kinase (ILK) signaling that was functionally confirmed by an ILK inhibitor. We suggest that cancer cells suppressed on SISgel could serve as a model for dormancy and re-awakening to allow for the identification of therapeutic targets for treating micrometastases.

Purpose The urothelium of cats diagnosed with feline interstitial cystitis (FIC) was analyzed to determine if abnormalities in protein expression patterns could be detected, and whether the pattern of expression was similar to that observed in human Interstitial Cystitis/Bladder Pain Syndrome (IC) patients. The proteins that were analyzed are involved in cell adhesion, barrier function, comprise the glycosaminoglycan (GAG) layer, or are markers of differentiation. Methods Formalin-fixed biopsies from 8 cats with FIC and 7 healthy controls were labeled using immunohistochemistry and scored using a modified version of a system previously used for human samples. Cluster analysis was performed to investigate relationships between the markers and samples. Results The results showed that 89% of the FIC bladders displayed abnormal protein expression and chondroitin sulfate (CS) patterns, whereas only 27% of the normal tissues exhibited slight abnormalities. Abnormalities were found in most of the FIC samples, biglycan (87.5%), CS (100%), decorin (100%), E-cadherin (100%), keratin-20 (K20, 100%), uroplakin (50%), ZO-1 (87.5%). In the FIC bladders, about 75% of the CS, biglycan, and decorin samples displayed absence of luminal staining or no staining. Results from the cluster analysis revealed that the FIC and normal samples fell into two clearly separate groups, demonstrating that the urothelium of cats with FIC is altered from normal. Conclusions FIC produces similar changes in luminal GAG and several proteins as is seen in human patients, suggesting some commonality in mechanism and supporting the use of FIC as a model for human IC.