Despite the molecular and histological similarities between fetal bone development and fracture healing, inflammation is an early phase of fracture healing that does not occur during development. Cyclo-oxygenase 2 (COX-2) is induced at inflammation sites and produces proinflammatory prostaglandins. To determine if COX-2 functions in fracture healing, rats were treated with COX-2-selective nonsteroidal anti-inflammatory drugs (NSAIDs) to stop COX-2-dependent prostaglandin production. Radiographic, histological, and mechanical testing determined that fracture healing failed in rats treated with COX-2-selective NSAIDs (celecoxib and rofecoxib). Normal fracture healing also failed in mice homozygous for a null mutation in the COX-2 gene. This shows that COX-2 activity is necessary for normal fracture healing and confirms that the effects of COX-2-selective NSAIDs on fracture healing is caused by inhibition of COX-2 activity and not from a drug side effect. Histological observations suggest that COX-2 is required for normal endochondral ossification during fracture healing. Because mice lacking Cox2 form normal skeletons, our observations indicate that fetal bone development and fracture healing are different and that COX-2 function is specifically essential for fracture healing.
Nephrons develop from mesenchymal cells that have contacted the ureteric bud (UB). To determine whether cell associated or secreted ureteric molecules induce the mesenchyme, we have isolated UB cell lines from mice transgenic for T antigen. These cells express epithelial and ureteric (Dolichos lectin staining, c-ret, c-met without hepatocyte growth factor) specific markers, which identifies them as authentic UB cells. Medium conditioned by our cells rescues mesenchyme from apoptosis without inducing the appearance of epithelial aggregates. The same was found by culturing mesenchymes upon the apical surface of a UB monolayer. In contrast, tubules were induced in mesenchymes contacting trypsinized pellets of UB cells. As revealed by staining for T antigen and Dolichos lectin or by prelabeling UB cells with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), we found that our cells encapsulated the mesenchyme but did not incorporate in the tubules. These data demonstrate that nephrogenesis is stimulated by two distinct ureteric signals, secreted molecules rescue the mesenchyme from apoptosis, whereas diffusion-limited basolateral molecules trigger mesenchymal/epithelial conversion.
Androgen deprivation subsequent to castration of an adult male rat results in the regression of sexual accessory tissues. Regression of these tissues involves the massive death of androgen-dependent cells. Using the rat ventral prostate gland as a model to study androgen-programed cell death, we have characterized a series of molecular events that accompany its regression. This analysis has shown that there was a sequential induction of specific gene transcripts in the ventral prostate gland following castration. The first event in this cascade was an abrupt induction of transcripts encoding c-fos. Since c-fos expression has been linked to perturbations in intracellular Ca2+ levels, we investigated whether membrane-mediated Ca2+ flux might be an early physiological step involved in the death of prostatic cells. To test this, rats were treated simultaneously upon castration with either verapamil or nifedipine, two different calcium channel antagonist drugs. Compared to the ventral prostate glands of untreated castrated rats, the glands of the calcium channel antagonist-treated rats showed a significant delay in all parameters associated with regression (loss of wet weight and DNA content and delay in histological changes associated with prostatic regression). The seminal vesicle glands of treated rats also showed signs of delayed regression. Furthermore, calcium channel antagonists suppressed the induction of transcripts encoding both c-fos and testosterone-repressed prostate message-2 (TRPM-2), a gene expressed exclusively by dying cells, during the first 62 hr following castration. These findings further support a role for calcium ion influx in the pathway leading to hormonally programed prostate cell death, and suggest the intriguing possibility that modulation of this activity can alter the process by which cells die.
SummaryThis study explored the use of interleukin 2 (I1,2) and interferon ~/(IFN-3') gene-modified tumor cells as cellular vaccines for the treatment of bladder cancer. The mouse MBT-2 tumor used is an excellent model for human bladder cancer. This carcinogen-induced tumor of bladder origin resembles human bladder cancer in its etiology and histology, and responds to treatment in a manner similar to its human counterpart. Using retroviral vectors, the human I1,2 and mouse IFN-qr genes were introduced and expressed in MBT-2 cells. The tumor-forming capacity of the cytokine gene--modified MBT-2 cells was significantly impaired, since no tumors formed in mice injected intradermaUy with either II.2-or IFN-y-secreting cells, using cell doses far exceeding the minimal tumorigenic dose of parental MBT-2 cells. Furthermore, mice that rejected the IL-2-or IFN-3,-secreting tumor cells became highly resistant to a subsequent challenge with parental MBT-2 cells, but not to 38C13 cells, a B cell lymphoma of the same genetic background. To approximate the conditions as closely as possible to the conditions prevailing in the cancer patient, inactivated cytokine-secreting cells were used to treat animals bearing tumors established by orthotopic implantation of MBT-2 cells into the bladder wall of the animal. Treatment of mice carrying a significant tumor burden with I1.2-secreting MBT-2 cells had a significant inhibitory effect on tumor progression with extended survival. Moreover, in 60% of the mice the tumor regressed completely and the animals remained alive and free of detectable tumor for the duration of the observation period. Treatment of tumor-bearing animals with I1.2-secreting MBT-2 cells was superior to the use of cisplatin, a chemotherapeutic agent used in the treatment of bladder cancer. The therapeutic effect of IFN-3,-secreting cells was minimal and treatment with unmodified MBT-2 cells had no effect on tumor growth or survival, showing that the parental MBT-2 cells were nonimmunogenic in this experimental setting. Most importantly, mice that exhibited complete tumor regression after treatment with I1.2-secreting MBT-2 cells became resistant to a subsequent challenge with a highly tumorigenic dose of parental MBT-2 cells, indicating that long-term immunological memory was established in the "cured" mice.
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