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.

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.

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.

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.

Learning from nudity: lessons from the nude phenotype.

In mice, rats, and humans, loss of function of Foxn1, a member of the winged helix/forkhead family of transcription factors, leads to macroscopic nudity and an inborn dysgenesis of the thymus. Nude (Foxn1(nu)/Foxn1(nu)) mice develop largely normal hair follicles and produce hair shafts. However, presumably because of a lack of certain hair keratins, the hair shafts that are generated twist and coil in the hair follicle infundibulum, which becomes dilated. Since hair shafts fail to penetrate the epidermis, macroscopic nudity results and generates the - grossly misleading - impression that nude mice are hairless. Here, we provide an overview of what is known on the role of Foxn1 in mammalian skin biology, its expression patterns in the hair follicle, its influence on hair follicle function, and onychocyte differentiation. We focus on the mechanisms and signaling pathways by which Foxn1 modulates keratinocyte differentiation in the hair follicle and nail apparatus and summarize the current knowledge on the molecular and functional consequences of a loss of function of the Foxn1 protein in skin. Foxn1 target genes, gene regulation of Foxn, and pharmacological manipulation of the nude phenotype (e.g. by cyclosporine A, KGF, and vitamin D3) are discussed, and important open questions as well as promising research strategies in Foxn1 biology are defined. Taken together, this review aims at delineating why enhanced research efforts in this comparatively neglected field of investigative dermatology promise important new insights into the controls of epithelial differentiation in mammalian skin.

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.

Histone deacetylase inhibitor, FK228, induces apoptosis and suppresses cell proliferation of human glioblastoma cells in vitro and in vivo.

We investigated the effects of FK228 on cell proliferation and apoptosis against human glioblastoma (GM) T98G, U251MG, and U87MG cells. Upon exposure to FK228, cell proliferation was inhibited, and apoptosis detected by the cleavage of CPP32 was induced. FK228 increased the expression levels of p21 (WAF-1) and of pro-apoptotic Bad protein in all GM cells. Furthermore, FK228 treatment also reduced the anti-apoptotic protein Bcl-xL in all GM cells and anti-apoptotic Bcl-2 in U87MG cells, thereby shifting the cellular equilibrium from life to death. An increased accumulation of histone H4 was detected in the p21 (WAF-1) promoter and the structural gene (exon 2) and the Bad structural gene (exon 2 and 3) upon treatment with FK228, as assessed by chromatin immunoprecipitation (ChIP) assay. Thus, the results indicated that an increased expression of p21 (WAF1) and Bad due to FK228 is regulated, at least in part, by the degree of acetylation of the gene-associated histone. We also found that FK228 inhibits cellular invasiveness and decreases MMP-2 activity. In addition, the growth of transplanted human GM m-3 cells into the subcutaneous tissue of hereditary athymic mice was significantly inhibited, and apoptosis was induced with FK228 treatment. The results suggested that FK228 might be useful in the treatment of human GM, although further studies will be needed.

Complete rescue of the nude mutant phenotype by a wild-type Foxn1 transgene.

In this paper we describe the production and analysis of mice carrying a 110-kb transgene that encompasses the wild-type Foxn1 genomic locus. Mutations in Foxn1 cause the nude phenotype. We show that in the hair follicles, transgenic mice with increased Foxn1 gene dosage exhibited increased Foxn1 expression that was restricted correctly to the nascent, post-mitotic cells of the differentiating hair cortex and hair cuticle lineages. We also demonstrate for the first time that a Foxn1 transgene rescues completely both the hair follicle and the thymus defects in animals that are also homozygous for the nude mutation at the endogenous Foxn1 locus, causing the development of a full coat of hair and a normal population of peripheral blood T lymphocytes. We conclude that sufficient cis-acting regulatory information resides within this 110-kb transgene to direct reliable and appropriate tissue-specific expression of the Foxn1 gene.

Adenoviral vectors stimulate murine natural killer cell responses and demonstrate antitumor activities in the absence of transgene expression.

Adenoviral vector-mediated gene delivery is currently the focus of many efforts to administer therapeutic gene products for the treatment of cancer. Although these vectors are replication deficient, they can induce specific immune responses against both vector- and transgene-encoded proteins. We have extended these findings to determine the level of innate natural killer (NK) cell responses to adenoviral vector administration in vivo. Similar to many replicating viruses, the vectors induce prominent NK cell activation in mouse spleens within 2 days of injection. We also observed these NK cell responses regardless of the route of administration. Furthermore, stimulation of NK cells by adenoviral vectors is independent of viral gene transcription, as UV inactivation of the vectors does not reduce the NK cell response. In contrast, heat treatment of the vectors destroys their ability to activate NK cells, demonstrating the necessity for intact vector particles. In addition, we found that administration of "empty" (no transgene) adenoviral vectors delays tumor growth in mice bearing B16 melanomas, and this effect is abrogated by depletion of NK cells. Collectively, these results demonstrate in a murine system that the adenoviral vector gene delivery system itself stimulates NK cells, and this in turn can nonspecifically enhance antitumor immunity.

Hair growth pattern in nude mice.

Nude mice are not bald but instead show an 'abortive' reduced hair growth on different sites of the integument. An albino (NMRI-nu) and a pigmented (C57BL/6-nu) strain of nude mice were examined as to whether the regional distribution pattern of this anagen hair proliferation is subject to the same ontogenetic development as in hairy mice. Hairy mice of both strains served as a comparison. Hair distribution was documented macroscopically by drawing and photography in a total of 415 mice of both sexes up to 421 days of age. Because of the pigmentation of the growing anagen hair follicles, the growth areas in the pigmented nude mice were distinctly visible whereas in the albino mice they were roughly recognisable from the boundaries of hair covering. The regional distribution of the 'abortive' anagen hair pattern in both nude strains corresponded to the wave-like course of the adult hair generations of hairy mice. As in older hairy mice, the hair cycle duration in nude mice was prolonged from an age of 121-180 days, the hair growth areas appeared reduced and less symmetrically orientated. Differences of up to 33% in body mass between the lighter nude and +/nu mice made ontogenetic comparison impossible so that all information is based on direct pattern or age comparison. The significance of experiments on the skin and hair follicles of nude mice is further increased if litters are examined comparatively and the temporal and spatial dimension of the follicle proliferation is considered more carefully than has been the case until now.

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%).

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.

Altered thyrotropic and somatotropic responses to environmental challenges in congenitally athymic mice.

Neonatal thymectomy or congenital absence of the thymus induces morphologic alterations in pituitary somatotrophs as well as in thyroid epithelium. It was therefore of interest to assess somatotropic and thyrotropic cell morphology and the corresponding serum hormone levels in athymic nude mice under basal and stressful conditions, taking as a reference their haired counterparts. Normal (+/+), heterozygous nude (nul+) and homozygous (nu/nu) CD-1 mice were subjected to either 1-h immobilization stress or 2-h cold stress. Serum levels of growth hormone (GH), thyrotropin (TSH), thyroxine (T4), and triiodothyronine (T3) were assessed by RIA at 0, 30, and 60 min poststress. Athymic animals showed lower basal levels of serum TSH, GH, and T3, but not T4, than their heterozygous littermates. Immunohistochemical assessment of somatotropic and thyrotropic cell populations revealed a normal morphology in the athymic animals. Immobilization stress induced a marked reduction in GH and TSH levels in normal mice but had only a weak effect in athymic animals. Two hours of cold exposure caused a comparable increase in serum TSH in normal and athymic animals, whereas the serum T4 and T3 response to cold was greater in the athymic nudes. Cold exposure drastically reduced serum GH levels in normal animals but had only a weak effect in the athymic mice. We conclude that congenital athymia in the mouse is associated with decreased basal levels of serum TSH and GH in the presence of a normal somatotroph and thyrotroph morphology. The anomalous responses of athymic mice to stress do not appear to be due to primary hypopituitarism but rather, to an altered modulation of pituitary hormone secretion.

Function of bone marrow stromal cell lines derived from nude mice.

We previously reported that in double deficient nude.xid mice B cells failed to develop and their bone marrow did not produce mature B cells in vitro. However, when progenitors from nude.xid bone marrow were placed on a preestablished normal stromal cell line (AC6) they differentiated into surface IgM+ cells. This raised the possibility of a deficiency of nude and nude.xid stromal cells such that they were incapable of supporting the maturation of X-linked immune deficiency (xid) B cells. Here we ask whether bone marrow stromal cells from nude and nude.xid mice have the ability to support xid B cell lymphopoiesis. A primary stromal cell layer derived from nude mice supported xid B cell differentiation in vitro. We derived panels of stromal cell lines by transfection of primary stromal cell layers with a retrovirus encoding SV40 large T Ag. Several bone marrow stromal cell lines derived from nude and nude.xid mice supported xid B cell differentiation from CD43+/CD45 (B220-) to CD45 (B220+) and from CD45 (B220+)/surface IgM- to surface IgM+. Supporting cell lines expressed both IL-7 and insulin-like growth factor I. The frequencies of bone marrow stromal cells capable of supporting xid B cell differentiation were similar in normal, xid, nude, and nude.xid mice. These results demonstrate that nude and nude.xid mice have bone marrow stromal cells with normal abilities to support B cell maturation.

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.

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.

Comparative abilities of athymic nude mice and severe combined immune deficient (SCID) mice to accept transplants of induced rat mammary carcinomas: enhanced transplantation efficiency of those rat mammary carcinomas that have elevated expression of neu oncogene.

Grafts of primary carcinogen (DMBA)-induced mammary carcinomas from Sprague-Dawley rats have a poor transplantation efficiency in athymic nude mice. Further compromising these mice immunologically via whole-body irradiation and/or splenectomy, or the administration of hormonal growth factors (estrogen and progesterone) to these mice, did not significantly alter transplantation efficiency. Use of strains of mice that are more immune-impaired than the athymic nude mouse, i.e., the athymic nude-beige-XID mouse (T-cell and LAK-cell deficient) or mice with severe combined immune deficiency (SCID) (which lack functional T cells and B cells) also failed to improve transplantation efficiency. In contrast, transplantation efficiency was sharply increased when primary neu-induced rat mammary carcinomas from female Sprague-Dawley rats were used. These mammary carcinomas, unlike the DMBA-induced rat mammary carcinomas, have a very high level of expression of neu; transplantation of these tumors to either athymic nude mice or SCID mice was considerably more efficient. Thus, these data provide evidence that enhanced expression of neu confers heightened efficiency in the transplantation of primary rat mammary carcinomas to immune-deficient mice (athymic-nude or SCID). Increased neu expression was a greater determinant than more compromised immune states in the transplantation of these rat mammary carcinomas. This biological characteristic of neu expression in mammary carcinomas provides new, additional insight into the importance of this oncogene in mammary tumorigenic processes and may explain, at least in part, the reported inverse relationship between human breast carcinoma neu expression and patient prognosis.

Spontaneous evolution of nuclear size and DNA content of non-small-cell-lung cancers grafted onto nude mice.

We monitored the biological evolution of four human non-small-cell lung cancers labeled KLX6, KLX7, KLX9, and KLX14 that had been grafted onto nude mice. This monitoring was carried out by means of digital cell image analysis which assessed morphometric (nuclear area, NA) and densitometric (nuclear DNA content, DI) features on Feulgen-stained nuclei from imprint smears. In each of the four models, the biological evolution of the NA and DI was characterized through their progression during serial passaging onto nude mice. The results show that of the four lung cancer models analyzed, one (KLX6) remained definitively stable with respect to its DNA content (DI assessments), while the other three, i.e., KLX7, KLX9, and KLX14, varied significantly. As assessed by the morphometric parameter, i.e., the NA, the four xenografted lines also varied significantly over serial passaging. In conclusion, we show that digital cell image analyses of Feulgen-stained cell nuclei including morphometric and densitometric parameters are a powerful tool for monitoring the biological evolution of human lung cancers grafted onto nude mice. We think therefore that the ploidy level of a tumor might be dependent upon its age and/or its related clinical stage.