Design and implementation of a novel superfusion system for ex vivo characterization of neural tissue by dielectric spectroscopy (DS).

Dielectric spectroscopy is a widely utilized electrophysiological characterization method. The obtained dielectric spectra and derived properties have the potential of providing significant information regarding changes in the physiological state of a biological system. However, since many of the dielectric properties are obtained in vitro from excised tissue far removed from physiological conditions, the value of the information obtained may be diminished. In this paper, we introduce a superfusion system that is designed to produce ex vivo dielectric spectroscopy measurements by providing the living tissue with a continuous and ample supply of nutrients and oxygen while removing metabolites and other waste. This superfusion system provides the convenience of in vitro measurement while concurrently producing results that can be more closely correlated with actual physiological changes in the biological system.

Dietary 4-HPR suppresses the development of bone metastasis in vivo in a mouse model of prostate cancer progression.

The effects of the synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) on prostate cancer metastasis in vivo were evaluated in the mouse prostate reconstitution (MPR) model. MPRs were produced by infection of either heterozygous (+/-) or nullizygous (-/-) p53-mutant fetal prostatic epithelial cells with the recombinant retrovirus Zipras/myc 9. Previous studies have documented that loss of p53 function potentiates metastasis in this model system. MPRs were grafted into homozygous (+/+) p53 male mice, fed a 4-HPR containing diet or a control diet and maintained until the status of tumor progression dictated sacrifice. Under these experimental conditions, treatment with 4-HPR did not have a significant effect on primary tumor wet weight for either p53 +/- or p53 -/- MPRs. For, p53 +/- MPRs the animals fed the 4-HPR diet had a slight improvement in survival and a significant reduction in the number of mesenteric metastases (P = 0.0477, t-test). Notably, in p53 +/- MPRs the incidence of metastasis to lumbar spine and sternum was 92% in the control animals compared to 54% in the 4-HPR treated animals (P = 0.035, chi2-test). In p53 -/- MPRs there was a trend toward a reduction in the number of soft tissue metastases to lung and liver in the 4-HPR group relative to the control diet group and a statistically significant reduction in the incidence of metastasis to bone was demonstrated in that 50% of control animals versus 30% of 4-HPR treated p53 -/- animals harbored bone metastases (P = 0 < 0.05, chi2-test). Cell lines were established from portions of the primary tumor and from selected metastatic deposits in each experimental group. Clonal analysis, by retroviral integration pattern, indicated increased clonal diversity in both the primary tumors and metastasis-derived cell lines from 4-HPR treated animals relative to the control animals. In vitro treatment with 4-HPR did not reveal discriminating differences between cell lines derived from primary tumors and bone metastases or control and treatment groups in regard to growth arrest or apoptotic responses. Overall these studies indicate limited anti-tumor and anti-metastatic activity in this highly aggressive in vivo mouse model of prostate cancer, yet 4-HPR treatment significantly suppressed the development of bone metastases in p53 +/- and p53 -/- MPRs revealing a novel and potentially clinically useful activity of this retinoid.

Retention of p53val135 wild-type function in transgenic mice.

We targeted a mutant p53 gene (val135), previously shown to cause tumors in transgenic mice, to the kidney and eye using a gamma-glutamyltranspeptidase promoter. Although transgene RNA was expressed in both tissues, and mutant protein could be detected at high levels in the kidney and was appropriately localized to the nuclei of proximal tubules, no gross or microscopic lesions developed, even when mice were held as long as 75 weeks. When these mice were crossed with transgenic mice carrying HrasT24 (containing a codon 12 mutation) driven by the same promoter, the p53val135 transgene partially suppressed the mutant ras phenotype (proximal tubular hyperplasia and adenomas and carcinomas of the ciliary body and retinal pigment epithelium). The kidneys of double transgenic mice younger than 25 weeks showed less tubular hyperplasia and cystic change than littermates carrying gamma-glutamyltranspeptidase(I)rasT24 alone. By 33 weeks, there was no difference in the severity of the kidney lesions. The eye lesions were less aggressive, and no malignant lesions were identified. Our findings are consistent with the work of others, indicating that p53val135 is not tumorigenic under all conditions; in fact, in some circumstances, it retains some of the suppressing activity of wild-type p53.

Transgenic mice carrying a PSArasT24 hybrid gene develop salivary gland and gastrointestinal tract neoplasms.

BACKGROUND: Although prostate cancer is one of the most prevalent tumors in men, knowledge of its biology has been hindered by lack of animal models. We have attempted to develop a prostate cancer model utilizing transgenic mouse technology. EXPERIMENTAL DESIGN: Two lines of transgenic mice were derived from one cell stage embryos injected with a fusion gene consisting of a mutated (codon 12) ras gene driven by the human prostate specific antigen (PSA) promoter in an attempt to target the oncogene specifically to the mouse prostate gland. Nontransgenic FVB/N mice were used as controls. The animals were sacrificed for study between 4 and 55 weeks of age. RESULTS: All organs were normal except the salivary glands and gastrointestinal tracts, both of which developed carcinomas in animals older than 44 weeks. The salivary gland tumors were of ductal origin, exhibited a variable degree of differentiation, and were shown to contain abundant PSAras mRNA by in situ hybridization. The gastrointestinal tract tumors were undifferentiated but appeared to be of stromal origin. Both salivary gland and gastrointestinal tumors occasionally metastasized. No transgene expression could be demonstrated in the prostate gland by either reverse transcription-polymerase chain reaction or in situ hybridization. CONCLUSIONS: Lack of transgene expression by the prostate can be explained on the basis of the apparent species specificity previously observed for PSA. Expression in salivary gland is best attributed to identity between the nucleotide sequences of the PSA promoter and of a mouse glandular kallikrein normally secreted by the salivary gland.

Evaluation of the chick wing territory as an equipotential self-differentiating system.

Harrison (1918: J. Exp. Zool. 25: 413-461) described a developmental field as an "equipotential self-differentiating system." The present study was undertaken to address the question: To what extent can be pre-limb territory of a chick embryo be considered a developmental field? To what extent is the chick pre-limb territory an equipotential self-differentiating system? Two sets of experiments were undertaken to address these questions: (1) Whole and half limb territories were explanted to the celoma of host embryos, and (2) portions of the wing territories were extirpated. The wing exhibited the quality of self-differentiation after stage 12, in that the isolated wing territory, grafted to a host celom, could form limbs beginning at stage 12 (however, complete wings formed only from wing territories of stage 16 and older). On the other hand, the chick wing territory did not appear to exhibit equipotentiality. No posterior half limb graft formed normal limbs, and only in two exceptional cases did anterior half limb grafts form limbs. If part or all of the wing territory was removed from chick embryos, normal limbs formed in less than 15% of the cases after stage 15, in about 30% of the cases at stages 13 and 14, but in over half the cases at stages 10-12. Wound healing and reinitiation of limb potential may be responsible for the higher incidence of limb formation at the younger ages.