Use of a murine xenograft model for canine transmissible venereal tumor.

OBJECTIVE: To develop a murine model for canine transmissible venereal tumor (CTVT). ANIMALS: Thirty-three 6-week-old NOD/LtSz-scid (NOD/SCID) mice and seven 6-week-old C57BL/6J mice. PROCEDURE: Samples of CTVT were excised from a 3-year-old dog and inoculated SC into ten 6-week-old NOD/SCID mice to induce growth of xenograft transmissible venereal tumor (XTVT). To establish mouse-to-mouse transmission, samples of XTVT were removed and inoculated SC into 4 groups of 6-week-old NOD/SCID mice and into a control group. Samples of CTVT were also inoculated into immunocompetent C57BL/6J mice for a mouse antibody production (MAP) test. The canine and xenografted tumors were evaluated cytologically and histologically, and polymerase chain reaction was performed for detection of the rearranged LINE/c-MYC junction. RESULTS: 8 of 10 NOD/SCID mice that were inoculated with CTVT developed tumors 3 to 10 weeks after inoculation. In the second-generation xenograft, all mice developed tumors by postinoculation day 47; 1 X 10(6) of XTVT cells were enough to create a xenograft. Metastases developed in 4 of 20 mice. Xenografted and metastatic tumors retained cytologic, histologic, and molecular characteristics of CTVT. Results of the MAP test were negative for all pathogens. CONCLUSION: We established an NOD/SCID murine model for XTVT and metastasis of CTVT. This model should facilitate study of tumor transplantation, progression, and metastasis and should decrease or eliminate the need for maintaining allogenic transfer in dogs.

Accentuated apoptosis in normally developing p53 knockout mouse embryos following genotoxic stress.

In order to identify the alternative pathways which may substitute for the p53 function during embryogenesis, we have focused our studies on p53 -/- normally developing mouse embryos that survived a genotoxic stress. We assumed that under these conditions p53-independent pathways, which physiologically control genomic stability, are enhanced. We found that while p53 +/+ mouse embryos elicited, as expected, a p53-dependent apoptosis, p53-/- normally developing mice exhibited an accentuated p53-independent apoptotic response. The p53-dependent apoptosis detected in p53+/+ embryos, was an immediate reaction mostly detected in the brain, whereas the p53-independent apoptosis was a delayed reaction with a prominent pattern observed in epithelial cells of most organs in the p53-deficient mice only. These results suggest that in the absence of p53-dependent apoptosis, which is a fast response to damaged DNA, p53-independent apoptotic pathways, with slower kinetics, are turned on to secure genome stability.

p53 tumor suppressor gene expression in the mouse ovary during an artificially induced ovulatory cycle.

OBJECTIVE: To evaluate the expression of p53 in the mouse ovary during an artificially induced ovulatory cycle. STUDY DESIGN: Ovulation induction was performed using pregnant mares' serum gonadotropin/human chorionic gonadotropin (PMSG/hCG). First, a p53 promoter-chloramphenicol acetyl transferase (CAT) transgenic mouse model was used. Protein samples from ovaries of transgenic mice were assayed for CAT activity as evidence of p53 promoter activation. Next, RNA extracted from CD-1 mouse ovaries was used for reverse transcription/polymerase chain reaction (PCR) and northern blot analysis using a p53-specific probe. RESULTS: Increased CAT activity was noted in transgenic mice treated with PMSG/hCG as compared with controls. PCR studies on transgenic mice using primers for CAT and on CD-1 mice using primers for wild type p53 substantiated this observation. Furthermore, CAT assay and northern analysis, performed on samples obtained at serial time intervals from induction, indicated that maximal p53 expression occurs around the time of ovulation, beginning 48 hours after PMSG and peaking 6-12 hours after hCG administration. CONCLUSION: The temporal expression of p53 in the ovary during a PMSG/hCG artificially induced ovulatory cycle may indicate a role for p53 in processes of differentiation of granulosa cells into luteal cells.

Does wild-type p53 play a role in normal cell differentiation?

Inactivation of the p53 tumor suppressor gene plays a major role in malignant transformation. The central question in this issue is concerned with the understanding of the function of p53 in normal cells and its deregulation in cancer cells. Several in vitro and in vivo experimental models have indicated that induction of cells to undergo differentiation involve up-regulation in the expression of the p53. In the case of B cell differentiation, p53 was found to be involved in several steps of the differentiation pathway. The conclusion that p53 plays a role in normal development and differentiation in vivo is substantiated by the observation that p53 is expressed during embryonic development and is detected at low levels in a number of organs of adult mice. Accentuated levels of p53 in testes of adult mice, suggests that p53 plays a role in the meiotic process of spermatogenesis. B cell differentiation and spermatogenesis are biological pathways which normally involve DNA reshuffling and rearrangements. In accordance with the notion that p53 is associated with DNA repair it is tempting to speculate that at least in these physiological pathways p53 functions as a master gene that controls genome integrity.

Mice with reduced levels of p53 protein exhibit the testicular giant-cell degenerative syndrome.

Transgenic mice which carry hybrid p53 promoter-chloramphenicol acetyltransferase (CAT) transgenes were found to express CAT enzymatic activity predominantly in the testes. Endogenous levels of p53 mRNA and protein were lower than in the nontransgenic control mice. The various p53 promoter-CAT transgenic mice exhibited in their testes multinucleated giant cells, a degenerative syndrome resulting presumably from the inability of the tetraploid primary spermatocytes to complete meiotic division. The giant-cell degenerative syndrome was also observed in some genetic strains of homozygous p53 null mice. In view of the hypothesis that p53 plays a role in DNA repair mechanisms, it is tempting to speculate that the physiological function of p53 that is specifically expressed in the meiotic pachytene phase of spermatogenesis is to allow adequate time for the DNA reshuffling and repair events which occur at this phase to be properly completed. Primary spermatocytes which have reduced p53 levels are probably impaired with respect to DNA repair, thus leading to the development of genetically defective giant cells that do not mature.

Testicular tissue-specific expression of the p53 suppressor gene.

The hybrid transgene approach was adapted to study the physiological pathway(s) in which the p53 suppressor gene is involved. p53 promoter-CAT transgenic mice were found to express enzymatic CAT activity predominantly in the testes. In situ hybridization indicated that expression of the transgene as well as the endogenous p53 agreed with the typical wave and cycle patterns of spermatogenesis. p53 promoter-CAT transgenic mice expressed in the testes reduced levels of endogenous p53 mRNA that correlated with the copy number of the mouse or human transgene. The spatial and cyclical expression of the p53 gene which is confined to the primary spermatocytes in the seminiferous tubuli suggested that p53 may play a role in the meiotic process of spermatogenesis in vivo.

Selecting, accelerating and suppressing interactions between macrophages and tumor cells.

Studies were carried out to test whether thioglycollate-induced macrophages (T-PM phi) exert a selective effect on 3LL tumor cells. One million 3LL tumor cells and 10 X 10(6) thioglycollate-elicited peritoneal macrophages were admixed and inoculated subcutaneously in C57BL/6 mice. This procedure was repeated for a series of 6-15 consecutive transplant generations. After 6 generations, the tumor cells which were selected in this manner (3LLR6) grew faster in vivo and in vitro when admixed with T-PM phi than 3LL cells which were not selected by T-PM phi. However, in vivo, this T-PM phi-mediated acceleration of 3LLR6 tumor growth was followed by tumor rejection and induction of anti-3LL immunity. Intravenous inoculation of T-PM phi enhanced the growth of pulmonary metastases in mice subsequently inoculated intravenously with 3LL cells. Such T-PM phi-mediated augmentation of metastases was not observed with 3LLR6 cells. Homing of 3LL and 3LLR6 cells to the lungs after pretreatment with T-PM phi was similar, indicating that these cells did not differ in their capacity to colonize the lungs but rather in subsequent tumor growth. Additional transplantations of 3LLR6 cells with T-PM phi led, after 9 further transplant generations, to a different tumor cell, 3LLR15, with completely new morphological, tumorigenic and metastatic properties. Unlike the 3LLR6 cells, this variant was not sensitive to the growth promoting effects of T-PM phi. Furthermore, it expressed the H-2K-and H-2D-encoded antigens of the H-2b haplotype, whereas the 3LL and 3LLR6 cells expressed only the H-2D alloantigens. This indicates that macrophages may either exert a selective effect on tumor cells or play a role in the induction of new tumor cells.