Sensitivity to carcinogenesis in nude mice: skin tumors caused by transplacental exposure to ethylnitrosourea.

Female athymic nude mice and their phenotypically normal littermates were exposed transplacentally to ethylnitrosourea. Skin tumors (papillomas and sebaceous adenomas) developed on the nude mice with an almost tenfold greater incidence than on their haired littermates. Skin tumors were also induced on nude mice but not haired controls by direct intraperitoneal treatment with ethylnitrosourea. These results indicate that nude mice have higher than normal susceptibility to carcinogenesis under some circumstances.

New method for detecting cellular transforming genes.

Tumor induction in athymic nude mice can be used to detect dominant transforming genes in cellular DNA. Mouse NIH 3T3 cells freshly transfected with either cloned Moloney sarcoma proviral DNA or cellular DNA's derived from virally transformed cells induced tumors when injected into athymic nu/nu mice. Tumors were also induced by cells transfected with DNA from two tumor-derived and one chemically transformed human cell lines. The mouse tumors induced by human cell line DNA's contained human DNA sequences, and DNA derived from these tumors was capable of inducing both tumors and foci on subsequent transfection. Tumor induction in nude mice represents a useful new method for the detection and selection of cells transformed by cellular oncogenes.

Functional barrel cortex in 'hairless', nude mice.

Although nude mice are not truly hairless, they demonstrate abnormal hair structure and growth patterns, which are related to their genetic state. Whereas wild-type mice are born with visible vibrissae, nude mice are distinguishable at birth by the lack of visible vibrissae, which do not appear until approximately postnatal day 6. Additionally, adult nude mice have abnormal whisker cycling patterns in which structurally normal whisker follicles produce fragile whiskers which break or fallout leaving follicles whiskerless for several days before a fine replacement whisker appears and develops. The current study shows that despite these abnormal periods of whisker deprivation, the barrel cortex of nude mice develops a normal structural appearance viewed with cytochrome oxidase staining. Additionally, intrinsic optical imaging studies of barrel cortex responses to single whisker stimulation do not appear altered from normal despite periodic loss of adjacent whiskers.

Athymic nude mice are insensitive to low-level toluene-induced up-regulation of memory-related gene expressions in the hippocampus.

The function of the N-methyl-d-aspartate (NMDA) subtype of glutamatergic receptors is known to be antagonized by toluene, a well-characterized neurotoxic chemical known to impair memory functions. Recently, peripheral T cells have been clearly shown to play an important role in cognitive and behavioral functions. In the present study, we investigated the role of peripheral T cells in the hippocampal mRNA expression of memory-related genes induced by low levels of toluene exposure in mice. BALB/c wild-type (WT) and nude mice were exposed to 9ppm of toluene or filtered air (0ppm toluene; control groups) in a nose-only exposure chamber for 30min on 3 consecutive days followed by weekly sessions for 4 weeks. Twenty-four hours after the last exposure, the hippocampi were collected and the inducibility of memory-related genes was examined using a real-time quantitative PCR method. NMDA NR2A, calcium/calmodulin-dependent protein kinase IV (CaMKIV), cyclic AMP-responsive element binding protein 1 (CREB1), and BDNF were significantly up-regulated in the hippocampi of WT mice exposed to 9ppm of toluene, compared to the expressions observed in WT mice exposed to filtered air, but similar results were not observed in nude mice. To investigate the possible involvement of peripheral T cells in the toluene-induced up-regulation of memory-related genes in WT mice, we examined the mRNA expression of Thy-1 (a pan T cell-specific marker) and quantified the number of cells that were immunoreactive to a T cell antigen receptor, CD3 (CD3-ir). Both the expression of Thy-1 mRNA and the number of CD3-ir cells were significantly higher in the hippocampi of the WT mice exposed to 9ppm of toluene, compared with that in WT mice exposed to filtered air; similar results were not observed in nude mice. We also examined the expression of chemokine genes like CCL2 and CCL3. The expression of CCL3 mRNA was significantly up-regulated only in the toluene-exposed WT mice. Although other differences unrelated to immune function may exist between WT and nude mice from the same background, the findings of the present study strongly suggest that the recruitment of peripheral T cells in the hippocampi of BALB/c WT mice exposed to low levels of toluene may be involved in the toluene-induced up-regulation of memory-related genes at the mRNA level.

Soluble bone-derived osteopontin promotes migration and stem-like behavior of breast cancer cells.

Breast cancer is a leading cause of cancer death in women, with the majority of these deaths caused by metastasis to distant organs. The most common site of breast cancer metastasis is the bone, which has been shown to provide a rich microenvironment that supports the migration and growth of breast cancer cells. Additionally, growing evidence suggests that breast cancer cells that do successfully metastasize have a stem-like phenotype including high activity of aldehyde dehydrogenase (ALDH) and/or a CD44+CD24- phenotype. In the current study, we tested the hypothesis that these ALDHhiCD44+CD24- breast cancer cells interact with factors in the bone secondary organ microenvironment to facilitate metastasis. Specifically, we focused on bone-derived osteopontin and its ability to promote the migration and stem-like phenotype of breast cancer cells. Our results indicate that bone-derived osteopontin promotes the migration, tumorsphere-forming ability and colony-forming ability of whole population and ALDHhiCD44+CD24- breast cancer cells in bone marrow-conditioned media (an ex vivo representation of the bone microenvironment) (p≤0.05). We also demonstrate that CD44 and RGD-dependent cell surface integrins facilitate this functional response to bone-derived osteopontin (p≤0.05), potentially through activation of WNK-1 and PRAS40-related pathways. Our findings suggest that soluble bone-derived osteopontin enhances the ability of breast cancer cells to migrate to the bone and maintain a stem-like phenotype within the bone microenvironment, and this may contribute to the establishment and growth of bone metastases.

Expression of metastatic potential of allogenic and xenogeneic neoplasms in young nude mice.

Several different mouse and rat tumors were injected s.c. or i.v. into specific-pathogen-free young (3-week-old) and adult (6-week-old) nude mice. All tumors grew s.c.; however, following i.v. injections, only metastatic neoplasms produced visible pulmonary tumor foci in the 3-week-old (but not in the 6-week-old) nude mice. The quantitative differences in metastatic potential among tumor lines and clones observed in syngeneic hosts were maintained also when 3-week-old nude mice were used. The enhanced survival of tumor cells in the lungs and the large numbers of visible pulmonary metastases observed in the young nude recipients correlated closely with the low levels of natural killer cell activity detected in 3-week-old (but not in 6-week-old) nude mice. In vivo activation of natural killer cells by treatment of mice with polyinosinic-polycytidylic acid or Corynebacterium parvum, 24 hr prior to tumor challenge rendered th 3-week-old nude mice resistant to development of metastases. We conclude that specific-pathogen-free 3-week-old nude mice, which lack functional T-lymphocytes and demonstrate low natural killer cell activity, could serve as an in vivo model to ascertain the metastatic potential of allogeneic and xenogeneic tumors.

Metastatic behavior of human tumor cell lines grown in the nude mouse.

The metastatic behavior of seven human tumor cell lines grown in young (3- to 4-week-old) nude mice was studied. Two cell lines were derived from malignant melanomas, one from a colon carcinoma, two from prostate adenocarcinomas, and two from renal adenocarcinomas. Many of the cell lines produced metastases after i.v. injection (experimental metastasis) and after s.c. transplantation (spontaneous metastasis) into young nude mice. The incidence of metastasis seemed dependent primarily on the biological characteristics of the individual tumor cell line. However, the incidence of metastasis of some tumor cell lines could be increased by isolation and establishment of variant sublines from secondary tumor deposits, by prolonged systemic administration of 17 beta-estradiol to suppress natural killer cell activity, and/or by use of an advantageous site of tumor implantation. Intrasplenic injection of tumor cells allowed the most dramatic overall expression of metastatic capacity in these cell lines, resulting in frequent and large metastases to liver, lungs, and the mesenteric, omental, and mediastinal lymph nodes.

Effect of Enrichment Devices on Aggression in Manipulated Nude Mice.

Agonistic behavior in group-housed male mice is a recurring problem in many animal research facilities. Common management procedures, such as the removal of aggressors, are moderately successful but often fail, owing to recurrence of aggressive behavior among cagemates. Studies have incorporated enrichment devices to attenuate aggression, but such devices have had mixed results. However, these studies did not include research manipulations when assessing the benefits of various enrichment devices. We obtained 100 male athymic nude mice and studied the efficacy of various enrichment devices, including cotton squares, paper rolls, shredded paper, nylon bones, and a mouse house and wheel combination in the reduction of fighting during an ongoing study that involved randomization followed by prostate and intratibial injections. Groups were evaluated according to a numerical grading system for wound assessment. Examination of the data revealed that the enrichment devices had no effect on the presence of wounds, thus none of the devices tested affected fighting in nude mice. However, when mice began experimental use, fight wounds increased significantly at cage change and after randomization, reflecting a disruption of existing social hierarchies. Therefore, in the context of an actual research study that involves common manipulations, the specific enrichment device had less effect on aggression in male nude mice than did the destruction and reconstruction of social structures within each group.

Use of a human skin-grafted nude mouse model for the evaluation of topical retinoic acid treatment.

Cultured human keratinocytes and artificial dermal equivalents maintained in vitro do not perfectly mimic the terminal differentiation patterns and response to drugs observed in intact human skin. We have made use of human skin grafted onto nude mice to demonstrate that such grafts maintain the pattern of pharmacologic responsiveness to all-trans retinoic acid previously reported in human subjects. The use of a quantitative polymerase chain reaction method to measure induction of a retinoic acid responsive gene, cytoplasmic retinoic acid binding protein II, has made it possible to generate objective data suitable for investigations of drug efficacy. This method of using grafted human skin has potential broad applicability for investigation of topical drugs in a number of therapeutic fields.

Hair growth cycles and wave patterns in "nude" mice.

Hair growth cycles and waves were studied through five generations of hair growth in C57BL/6Icr "nude" mice. One group of nudes received thymus grafts, a second group was composed of athymid nudes and a third consisted of heterozygous (nu/&) haired littermates. The results showed that hair growth cycles and wave patterns were essentially the same in thymus-restored nudes and athymic nudes which indicated that thymus did not play a role in these phenomena. The time interval between hair cycles was considerably shorter in both groups of nude mice as compared to heterozygotes (nu/&). Finally, the hair growth wave pattern in nude mice did not change throughout the generation of hair growth whereas profound changes in wave patterns were observed in heterozygous (nu/&) littermates.

Novel experimental models of human cancer metastasis in nude mice: lung metastasis, intraabdominal carcinomatosis with ascites, and liver metastasis.

The ability of RPMI 4788 cells, a human colon cancer cell line, to produce experimental metastases in the lung, intraperitoneal cavity, and liver was studied in nude mice. Injection of 2 X 10(6) tumor cells into the tail vein of nude mice produced metastatic lung tumors, and an intraportal injection of 5 X 10(6) cells produced metastatic liver tumors. An intraabdominal carcinomatosis with ascites was formed after an i.p. injection of 5 X 10(6) tumor cells. The nude mice with lung metastasis or intraabdominal carcinomatosis always died within a few weeks. Macroscopic observation showed that the number of lung metastatic nodules on day 21 after tumor inoculation was 311.3 +/- 78.2 (mean +/- SD) in BALB/C nude mice, and 187.5 +/- 26.7 in ICR nude mice. In survival experiments, the mice with intraabdominal carcinomatosis showed a mean survival of 29.0 +/- 1.7 (mean +/- SD) days in BALB/C nude mice and 43.6 +/- 6.1 days in ICR nude mice. These novel experimental models of metastases in nude mice produced by injection of RPMI4788 cells had high reproducibility and may be useful not only for the study of the metastatic process but also for testing anticancer drugs.

Function in athymic nude mice of parathyroid heterografts from patients with primary hyperparathyroidism and secondary hyperparathyroidism.

Heterotransplantation of adenomatous parathyroid glandular tissue from humans with primary hyperparathyroidism into athymic nude mice creates a unique animal model of this disease. The mice manifest high concentrations of both midregion/C-terminal human parathyroid hormone and biologically active intact human parathyroid hormone relative to either mice with no implants or mice that received normal human parathyroid tissue. Secretion of these substances is maintained in most mice for at least 9 to 13 months after implantation. In addition, animals that have experienced implantation exhibit other characteristics associated with human primary hyperparathyroidism including relative hypercalcemia and increased renal 25-hydroxyvitamin D-1 alpha-hydroxylase activity. We also measured these parameters in a group of nude mice that received transplantation of a similar mass of hyperplastic parathyroid tissue that was obtained from patients with uremic secondary hyperparathyroidism. Although we hypothesized that the level of human parathyroid hormone secretion from these implants would fall over time in response to the normal host environment, hormone levels remained as high as those in recipients of adenomatous heterografts, even after 9 to 13 months. Moreover, similar biologic effect of the excess parathyroid hormone (i.e., relative hypercalcemia, hyperphosphatasemia, and increased 1,25-dihydroxyvitamin D biosynthesis) were detected. These animal models should prove extremely useful in supplementing our understanding of hyperparathyroid disorder in man.

Lifespan of immunosuppressed NMRI-mice is increased by deprenyl.

Immunosuppressed NMRI-mice (nu/nu) were raised and kept under germ-reduced conditions and fed with a germ-reduced diet (14 animals = controls). For another 14 mice 4 mg of selegiline were admixed to 10 kg of the diet. The 50% survival rate of the latter group was 160% from birth or 220% from the beginning of the study. The survival rate in weeks finally reached 350%, and the area under the curve 250%. The last mouse in the control group died at the age of 5 months, 2.5 months after the study was started; the last mouse in the selegiline group died at the age of 14.5 months, 1 year after the beginning of the study.

Xenotransplantability of human squamous cell lung cancer in nude mice is not affected by angiogenic factors.

Sixty-two human squamous cell lung carcinomas were analyzed for expression of various angiogenic growth factors and their receptors using immunohistochemistry. The data were correlated with xenotransplantability of these tumors in nude mice. None of the factors investigated did show an association with xenotransplantability. However, there was a trend that specimens lacking VEGF165 were established as xenografts at a higher incidence (52%) than those expressing VEGF165 (39%).

IkappaBalphaM suppresses angiogenesis and tumorigenesis promoted by a constitutively active mutant EGFR in human glioma cells.

Human glioma cell lines (G36DeltaEGFR and IN500DeltaEGFR) have been shown to display an enhanced tumorigenic phenotype, when transfected with a constitutively active form of the epidermal growth factor receptor (DeltaEGFR). These cells were transfected with a mutant IkappaBalpha (IkappaBalphaM) that is resistant to phosphorylation and degradation, and hence blocks NF-kappaB activity. Recently, EGFR has been shown to increase the activity of NF-kappaB and to induce angiogenesis. In this report, we asked if IkappaBalphaM gene transfer into human glioma cell lines would inhibit tumorigenicity and angiogenesis in glioma. IkappaBalphaM inhibited in vitro and in vivo expression of vascular endothelial growth factor (VEGF) and interleukin 8 (IL-8). Human glioma xenografts treated with IkappaBalphaM gene transfer exhibited significantly decreased angiogenesis both in an orthotopic and in an ectopic model. The decreased expression of VEGF and IL-8 directly correlated with decreased tumorigenicity, and tumor vascularization. Taken in combination, these results provide strong evidence of IkappaBalphaM's role in regulating glioma angiogenesis even in the presence of constitutive EGFR activation.

Superoxide dismutase isoenzymes in tissues and plasma from New Zealand black mice, nude mice and normal BALB/c mice.

In various types of autoimmune disease, an increased frequency of spontaneous chromosome breaks has been reported. Plasma from such patients induces chromosome breaks in normal cells. Exposure of plasma to superoxide radicals increases the breakage activity, and addition of superoxide dismutase protects against it. The New Zealand black mouse is an animal model of autoimmune disease which displays the breakage phenomenon. To test for the possibility that the breakage is related to deficient protection against superoxide radicals, the activities of superoxide dismutase isoenzymes were determined in tissues and blood from New Zealand black mice and compared with the activities of normal BALB/c mice. No differences between the strains were revealed in tissue EC-superoxide dismutase, CuZn superoxide dismutase and Mn superoxide dismutase activity. The erythrocyte superoxide dismutase activities were also equal. The plasma EC-superoxide dismutase activity was 35% lower in the New Zealand black mice than in the BALB/c mice. Between euthymic BALB/c mice and nude mice, previously reported to be deficient in tissue superoxide dismutase activity, no difference could be demonstrated.

The influence of the athymic mutation nude on the components of the circadian rhythm of activity in mice.

The mutation known as nude brings about the lack of a thymus gland in mice. This immunodeficiency akes it possible to graft normally unaccepted, human cancerous tumors onto the mouse. Consequently, this animal is frequently used as a model for evaluating anti-cancer therapies. The effect of this mutation on biological rhythms constitutes a necessary step before using this model for cancer chronotherapy research. We evaluated the circadian and ultradian components of the rest-activity cycle in the following strains of mice: C57BL/6 with homozygous nu/nu, heterozygous nu/+, thymectomised +/+, and sham-operated +/+. The amount of activity was reduced in nu/nu as compared to the other groups. Nonetheless, neither the nude mutation nor thymectomy yielded any notable change in the circadian rhythm of activity.

Nude mouse models as predictors of chemotherapy in man: thymidine and pyrimidines.

The National Cancer Institute cancer treatment screening program has been reorganized incorporating, as an important feature, a panel of human tumors growing as xenografts in congenitally athymic mice. The new screening program is a prospective experiment in the search for new and more effective agents for the treatment of clinical neoplasia. The new program is described and questions that are being asked prospectively are presented. Data are summarized on the activity against human tumor xenografts for a number of clinically established antitumor drugs and examples are presented in which there is interest in compounds for the clinic on the basis of activity in the new screen. Studies are outlined in which high dose thymidine inhibited the growth of human melanoma and teratocarcinoma transplanted in athymic mice. Studies are discussed employing murine tumors in which marked augmentation of the in vivo antitumor activity of 5-fluorouracil was obtained by combination therapy with the pyrimidine nucleosides thymidine, uridine and cytidine. The desirability of investigating combination chemotherapy with pyrimidine nucleosides and 5-fluorouracil and other pyrimidine antagonists in the treatment of human tumor xenografts is stressed. There is a broad range of investigations that can be conducted in nude mouse models and it is important to conduct such programs in relation to the clinic.

Postnatal and postpartal morphology of the mammary gland in nude mice.

The object of this work was to compare the postnatal and postpartal morphology of the mammary gland of nu/nu with that of nu/(+)-mice. All studies were carried out on groups of female (athymic) nude mice with NMRI genetic background, their nu/(+)-siblings and dams. The various age groups (3, 21, 40, 55, 70 and 120 days) each consisted of 6 nu/nu- and 6 heterozygous nu/(+)-mice respectively. The morphological examination of the mammary gland tissue were made on histological sections and whole mounts. Body weights, total areas of the mammary glands and the number of the terminal end buds were compared. The mammary gland of the athymic nude mouse exhibited no essential morphological differences from the normal developing mammary gland of the hairy euthymic nu/(+)-animal. The area of the mammary gland increased with increasing body weight. Both collectives of mice differed only in their rate of mammary gland development. As a result, the terminal end buds appeared numerously as growth points of mammary gland in nu/(+)-animals as early as the 21st day of life. The athymic nude mice showed a maximum only on the 40th day of life and a lower degree of density and differentiation of specific mammary gland structures (lateral buds, lobulo-alveolar glandular endings) until the 70th day of life. The mammary gland of 120-day-old animals and dams of both animal groups reached the same state of maturity. Thus it is not the rate of development of the dam, but other, yet unidentified factors, which determine, if successful breeding of nude mice with homozygous parents is possible.

[Effect of the nude mutation on alpha-fetoprotein synthesis and hemopoiesis in the mouse liver in the postnatal period of development]. / Vliianie mutatsii nude na sintez al'fa-fetoproteina i gempoéz v pecheni myshei v postnatal'nom periode razvitiia.

The dynamics of population of alpha-fetoprotein (AFP)-containing cells in the liver and the level of AFP in the blood of C3H/HeJ+/+ and thymus-less mutant C3H/HeJnu/nu mice during postnatal development was studied by means of indirect immunofluorescence and radial immunodiffusion. The content of AFP-positive hepatocytes and AFP concentration in the blood serum of C3H/HeJnu/nu mice were shown to exceed markedly those in C3H/HeJ+/+ mice beginning from the age of 2 weeks. The histological analyses has revealed the foci of hemopoiesis in the liver of adult C3H/HeJnu/nu mice, unlike in the liver of normal mice. The neonatal thymectomy of C3H/HeJ+/+ mice did not influence the parameters under study. A possible relationship between the increased AFP level and the preservation of hemopoiesis in the liver of the mice homozygous by the mutation nude is discussed.