A novel methodology for analysis of cell distribution in chimeric mouse organs using a strain specific antibody.

Chimeric animals are very useful for analysis of cell lineage, homeostasis in tissue architecture, and cell-cell interactions during both organogenesis and carcinogenesis. However, there is not a generally effective means for marking cells of chimeric mice. We have therefore developed a polyclonal antibody that is useful for this purpose. This antibody specifically recognizes those cells derived from C3H strain mice. The specificity of this antibody was checked by both immunoblotting and immunoadsorption methods. The antigens were immunohistochemically detected in cytoplasm of both epithelial and mesenchymal cells of C3H/HeN strain mouse in many different organs, but not the corresponding cell types from BALB/c or C57BL/10 or several other mouse strains. The validity of these antibodies as markers for C3H cells was further checked by tissue recombination experiments and in mixed cultures of mouse and rat cells. In each case the antibody recognized only the C3H mouse cells. Next, chimeric mice were prepared between strains C3H/HeN and BALB/c, and C3H/HeN and C57BL/10 mice. Chimeras 2-mo old were examined for antigen distribution using the indirect immunofluorescence method. Many tissues in chimeric mice were composed of cells that were both stained and unstained by the anti-C3H specific antigen. The chimeric patterns were classified into four types, A-D. In well-defined structural units such as intestinal crypts, small intestinal villi, kidney convoluted tubules, exocrine gland acini, ovarian follicles, thyroid gland follicles, stomach glands, adrenal cortex, lingual papillae, etc., (A) each unit was composed entirely of either positive or negative cells, or else (B) in some organs each unit was composed of both types of cells. In the uniform tissues without such distinguishable units, such as stratified squamous epithelium, mesenchymal tissue, corpora lutea, pituitary gland, Islets of Langerhans, adrenal medulla etc., (C) the tissue was composed of definite small cell groups made entirely of either positive or negative cells, or else (D) the tissue was composed of both types of cells which were intermingled with one another. These findings strongly suggest that the chimeric patterns demonstrated here reflect the cell proliferative unit in each tissue. This cell marker system has proven useful for analysis of cell lineage and cell renewal systems in many organs of chimeric mice.

The distribution of tenascin-X is distinct and often reciprocal to that of tenascin-C.

We have isolated a cDNA encoding mouse tenascin-X (TN-X), a new member of the family of tenascin genes. The TN-X gene lies in the major histocompatibility complex (MHC) class III region, as it is the case for its human counterpart. On Northern blots we detected a TN-X mRNA of approximately 13 kb in most tissues analyzed, whereas in various mouse cell lines mRNAs of approximately 11 and 13 kb were detected, suggesting the possibility of alternative splicing of TN-X transcripts. We raised antibodies against mouse TN-X fragments expressed in bacteria and used these antibodies to identify the TN-X protein in heart cell extracts and in the conditioned medium of a renal carcinoma cell line. The subunit molecular size of TN-X is approximately 500 kD, suggesting that the protein may contain up to 40 fibronectin type III repeats, making it the largest tenascin family member known yet. TN-X in conditioned medium, as well as the purified protein bind to heparin, but no binding to tenascin-C (TN-C), fibronectin, laminin or collagens could be detected. Thus the heparin-binding activity may be a common feature of the tenascins. The TN-X mRNA as well as the protein are predominantly expressed in heart and skeletal muscle, but the mRNA is found in most tissues at a low level. Immunostaining showed the protein to be associated with the extracellular matrix of the muscle tissues and with blood vessels in all of the tissues analyzed. Although the TN-X gene lies in the MHC class III locus, it is not expressed in the lymphoid organs analyzed, except for the staining around blood vessels. In skin and tissues of the digestive tract often a reciprocal distribution of TN-X and TN-C was observed.

Thymus and reproduction: sex-linked dysgenesia of the gonad after neonatal thymectomy in mice.

Neonatal thymectomy of mice, when no ectopic thymus existed, constantly resulted in developmental arrest of the ovary but not of the testis; it also caused sterility in the female. The ovaries of thymectomized mice were extremely small and were characterized by absence of follicles and corpora lutea. Such an ovarian dysgenesia was observed when the mice were thymectomized at 3 days of age, but not at 7 days or later; it was prevented by thymus grafting.

Mesenchyme-dependent morphogenesis and epithelium-specific cytodifferentiation in mouse mammary gland.

Isografts of heterotypic recombinants of embryonic mammary epithelium with salivary mesenchyme undergo development morphogenetically resembling that of salivary gland. However, cytodifferentiation of the epithelium is like that of mammary gland. In lactating hosts these isografts respond to endogenous hormonal stimulation and synthesize a milk protein, alpha-lactalbumin.

Accelerated mammary cancer development by fetal salivary mesenchyma isografted to adult mouse mammary epithelium.

Transplantation of fetal salivary mesenchyma into adult mammary glands resulted in atypical outgrowths from the mammary duct system. These duct-alveolus nodules (DAN) were distinguishable from hyperplastic alveolar nodules (HAN) that arose from normal mammary duct systems in mice infected with murine mammary tumor virus (MuMTV). DAN displayed a type of ductal branching characteristic of salivary gland rather than of mammary gland, reflecting a tissue-specific perturbation of epithelium-mesenchyma in DAN in milk-transmitted MuMTV-infected C3H/HeN mice and in MuMTV-negative BALB/c mice given 7,12-dimethylbenz[a]anthracene (DMBA) subsequent to transplantation of fetal salivary mesenchyma. Mammary cancers were not increased in milk-transmitted MuMTV-free C3H/HeN and GRS/A mice that received salivary mesenchyma transplants. Salivary mesenchyma accelerated mammary carcinogenesis by increasing the mammary epithelial cell population responsive to MuMTV and DMBA.

Capacity of mammary fat pads of adult C3H/HeMs mice to interact morphogenetically with fetal mammary epithelium.

When rudimentary mammary epithelium from 13- to 17-day female C3H/HeMs fetuses was transplanted into gland-free mammary fat pads of 3-week-old mice, organogenetic development of the grafts occurred, resembling that seen in normal mammary gland morphogenesis. Initial developmental growth did not require the reproductive hormones. Mammary fat pads of juvenile (3-wk-old), young adult (8- to 12-wk-old), and fully matured (40-wk-old) females had equal ability to interact morphogenetically with fetal mammary epithelium. Fetal pulmonary, pancreatic, and salivary gland epithella showed no morphogenetic response within adult mammary fat. An exception was rudimentary hair follicle epithellum, which underwent extensive development toward hair follicles within mammary fat. Mammary glands that developed from rudimentary mammary epithellum transplanted into gland-free fat pads underwent morphologic changes characteristic of lactation when the hosts bore young.

Intestine-like remodeling of adult mouse glandular stomach by implanting of fetal intestinal mesenchyme.

A morphogenetic response of adult glandular stomach grown in contact with implanted fetal intestinal mesenchyme has been demonstrated. Mesenchymal tissues from intestines of 14- to 16-day-old BALB/c mouse fetuses were introduced beneath the epithelial layer of glandular stomach in 2-month-old mice and allowed to develop. Three to 4 weeks later, remodeling of the epithelial architecture had occurred; the characteristic glandular pit structure of normal stomach had been replaced by immature villi and crypts composed of mucus-secreting columnar cells more characteristic of intestinal tissues. Chief and parietal cells had disappeared, but neither goblet nor Paneth cells were observed. Such intestine-like morphogenesis was not induced by similarly implanted mesenchymal controls from fetal glandular stomach, forestomach, and salivary gland. A possible role of the mesenchymal stroma in the pathogenesis of intestinal metaplasia in stomach is discussed.

Strain specific sensitivity to diethylnitrosamine-induced carcinogenesis is maintained in hepatocytes of C3H/HeN in equilibrium with C57BL/6N chimeric mice.

The C3H/HeN (C3H) and C57BL/6N (C57) mouse strains are known, respectively, for their high and low susceptibility to both spontaneous and chemically induced hepatocarcinogenesis. The present study was aimed at elucidating whether this difference is dependent on intrinsic features of the target hepatocytes or in the in vivo milieu and associated growth promoting factors to which the cells are exposed. C3H in equilibrium with C57 chimeric mice were produced and given injections of diethylnitrosamine (20 microgram/g body weight) at the age of 15 days. The animals were sacrificed 6 or 9 months thereafter, and the numbers and sizes of altered cell lesions were scored. The clonal growth of both cell types was immunohistochemically confirmed using anti C3H-specific antigen antibodies. Quantitative assessment revealed C3H lesions in the chimera livers to be far larger (5:1) than those of C57 derivation and associated with more frequent malignant progression as was evident histologically. Furthermore, foamy change and hyalin body formation, which have been described as characteristics of C3H and C57BL hepatic tumors, respectively, were also featured as differentiative characteristics in lesions of both cell types in chimera mice. Thus, the results clearly demonstrated that the principal mechanism(s) underlying strain difference in diethylnitrosamine-initiated hepatocarcinogenesis exists in the target cells and is not milieu-dependent.

Tenascin expression in cancer cells and stroma of human breast cancer and its prognostic significance.

Sections of formalin-fixed, paraffin-embedded tissues from 210 human breast cancers were immunohistochemically examined using the mAb against human tenascin (TN) RCB1. Immunoreactive TN was detected in the breast cancer stroma in 77 (36.7%) cases, whereas the remaining 133 (63.3%) were negative. Of the 77, 12 (5.7%) cases also showed positive staining in the carcinoma cell cytoplasm. The positive cells were often observed in the margin of the cancer nests at the site adjacent to the stroma. According to the staining pattern of TN, the breast cancer cases were classified into the three groups of cancer cell TN(+)/stromal TN(+), cancer cell(-)/stromal TN(+), and cancer cell(-)/stromal TN(-). Analysis of the relationship of these TN patterns with various clinicopathological characteristics of the tumors and the patient outcome revealed that, in comparison to the cancer cell(-)/stromal TN(-) group, the cancer cell TN(+)/stromal TN(+) group exhibited increased frequency of lymph node metastasis and exceptionally poor outcome, and the cancer cell(-)/stromal TN(+) group also showed more frequent metastasis and poorer outcome. Most of the cancer cell TN(+)/stromal TN(+) cases were c-erbB-2 positive and estrogen receptor negative. Furthermore, in situ hybridization of freshly obtained breast cancer tissues demonstrated that both cancer cells and stromal cells express TN mRNA. These results indicate that the TN in breast cancer is produced by cancer epithelial cells as well as by stromal mesenchymal cells, and that cancer cell TN might be involved in cancer spreading, resulting in unfavorable patient prognosis.

Appearance of tenascin in healing skin of the mouse: possible involvement in seaming of wounded tissues.

Distribution of the extracellular matrix glycoprotein tenascin during wound healing in mouse skin was studied immunohistochemically. Within 24 hours after wounding, and preceding the formation of granulation tissue, tenascin appeared in the basement membranes beneath epidermis and hair follicles adjacent to the wound edges and in the wounded edges of cutaneous muscle layer. Granulation tissue began to form in the wound space at about 1-2 days and was immediately covered by epidermis. Tenascin first appeared in the periphery of the granulation tissue beneath healing epidermis and around the wounded edges of cutaneous muscle layer. Then the tenascin-positive area extended into the inner region of granulation tissue. At about 5-7 days, all of the granulation tissue was intensely stained with anti-tenascin serum. Tenascin immunoreactivity decreased as granulation tissue was replaced with reconstructed dermal tissue at 7-14 days. In most cases, tenascin staining persisted longest in the dermis beneath the healing epidermis and at the juncture of healing edges of cutaneous muscle layer. It disappeared at about 10-14 days after wounding. These findings suggest that tenascin may play an important role in the seaming of wounded tissues.

Analysis of tenascin mRNA expression in the murine mammary gland from embryogenesis to carcinogenesis: an in situ hybridization study.

The expression of tenascin gene during murine mammary gland development was analyzed by in situ hybridization with non-radioactive cRNA probes. The aim was to identify whether cells that synthesize tenascin are mesenchymal or epithelial. During embryogenesis, tenascin mRNAs were demonstrated in the epithelial cells of the mammary bud on the 14th and 15th day of gestation, and in the mesenchymal cells from the 14th day to the 17th day, at the epithelial-mesenchymal border of the growing bud. However, cells displaying tenascin mRNAs were not found beyond the bifurcation of the mammary sprout at the beginning of the branching morphogenesis. In post-natal development, tenascin mRNAs were demonstrated in mesenchymal cells surrounding end buds in juvenile mice, in mesenchymal cells surrounding the epithelial cells of plaques, in epithelial cells of the lactating mammary gland, in malignant epithelial cells and in the mesenchymal cells surrounding cancer nests. By immunohistochemistry, tenascin immunoreactivity was shown to have the same spatiotemporal distribution as that of tenascin mRNAs, but was observed to be restricted to the stroma, except in the lactating mammary gland where tenascin was demonstrated in the milk by Western blot. The present study thus showed that both epithelial and mesenchymal cells are sources of tenascin at different stages of murine mammary gland development.

Tenascin expression and postnatal development of the human prostate.

We investigated the distribution of tenascin during postnatal development of the human prostate. Monoclonal antibody specific to human tenascin was applied to paraffin sections by the avidin-biotin-complex method to examine its localization. Infantile prostates showed two distinct zones. The inner zone had sparse acini with fibromuscular bundles. The peripheral zone had similar acini and lighter stroma as compared with inner zone. Tenascin was found diffusely but weakly. The periglandular area occasionally showed immunoreactivity. The prostates from 9- to 13-year-old subjects had a morphology similar to that of the infantile gland. The difference was increased density of the acini. Immunoreactivity was low. In the prostates from 14- to 21-year-old subjects, the acini distribution was more crowded, and the epithelial lining had become taller in both zones. Tenascin distributed preferentially in the peripheral zone during this period. Simultaneously, the percent of glandular area in the peripheral zone rose abruptly. The dynamics of tenascin expression are closely associated with the development and maturation of the gland. The distribution of tenascin during the post-puberal period may suggest its participation in the preferential of prostatic carcinoma in the peripheral zone.

Isolation and characterization of human fibroblast tenascin. An extracellular matrix glycoprotein of interest for developmental studies.

We have developed a biochemical method for purifying human tenascin from cultured fibroblasts or the culture medium. The method is a series of biochemical procedures including gel filtration, gelatin gel affinity chromatography and ion-exchange high performance liquid chromatography. The final preparation was identified as tenascin from its immunological cross-reactivity to antibody against chicken tenascin, strong hemagglutination activity which has been reported to be one of the biological functions of chicken tenascin, and from the electron microscopic study demonstrating a six-armed structure. Gel chromatography showed that intact human tenascin has an apparent molecular weight of over one million. Analysis of the purified tenascin with SDS-PAGE under reducing conditions demonstrated that tenascin consists of two kinds of subunits (250K and 190K). We established rat x mouse heterohybridoma cell lines which produce tenascin-specific antibodies. One monoclonal antibody (RCB1) was selected for immunohistochemical study and partially characterized. RCB1 bound native tenascin but not reduced and alkylated tenascin. Immunohistochemistry of normal and neoplastic tissues demonstrated that RCB1 bound the connective tissues surrounding the cancer nests and various normal tissues including interstitium of renal distal tubule, periosteum, endosteum, smooth muscles of digestive tract and media of arteries and arterioles.

Murine tenascin: cDNA cloning, structure and temporal expression of isoforms.

Mouse tenascin (TN)-encoding cDNA clones were isolated from a cDNA library of the 2H6GR mammary tumor cell line. Nucleotide (nt) and deduced amino acid (aa) sequences revealed the characteristic primary structure, which begins with a signal peptide and TN unique sequences, follows with 14 1/2 epidermal growth factor (EGF)-like repeats and 13 fibronectin type-III repeats (FN repeat), and concludes with fibrinogen-homologous sequences. Similar to chicken and human TN, the mouse TN cDNA contains five consecutive insertional FN repeats, as well as eight constitutive FN repeats. Three different cDNA clones that may have been generated by alternative splicing of these insertional FN repeats were identified and characterized. Based upon the deduced as sequence, a polyclonal antibody was produced against a synthetic TN peptide. It specifically recognized two TN isoforms of 230 kDNA and 190 kDa in protein extracts of mouse tissues. The tissue distributions of mouse TN mRNAs, revealed by Northern blot analysis, suggest that there is tissue-specific expression of TN isoforms. Two distinct mRNA transcripts (7 kb and 5.5 kg) were detected in brain, skeletal muscle, digestive tract and bladder, but only one was observed in lung, kidney (7 kg) and thymus (5.5 kg). TN mRNA expression was down-regulated 1 month after birth in most tissues. However, the 5.5-kb transcript persisted in cerebellum, thymus, and colon. The spatial and temporal patterns of TN expression seem to be controlled at the level of transcription, because analysis of various tissues by Western blots showed the same pattern as that seen in Northern blots.

Structure and organization of the gene encoding a mouse mitochondrial stress-70 protein.

We have previously found that an antigenic protein specific for C3H strain mouse (C3H strain-specific antigen, CSA) is identical to peptide-binding protein 74 (PBP74). PBP74/CSA is a novel member of the stress-70 protein family in mitochondria. In this study, mouse genomic clones encoding PBP74/CSA, including the 5'- and 3'-flanking regions of the gene, have been isolated and sequenced. The PBP74/CSA gene contained 17 exons interrupted by 16 introns. Two dimeric repeats of the consensus sequence of the heat-shock element are present in the 5'-flanking region of the PBP74/CSA gene. Moreover, the first intron is interrupted within the amino-terminal leader sequence, the pattern of which is similar to that of cytochrome c1 located in the mitochondria.

Susceptibility of tenascin to degradation by matrix metalloproteinases and serine proteinases.

The degradation of tenascin purified from human melanoma cells was examined by treatment with matrix metalloproteinases (MMPs) and serine proteinases. Among eight different types of proteinases examined, MMP-1, -3, and -7, cathepsin G and leukocyte elastase could digest tenascin, but MMP-2, MMP-9 and thrombin did not. This suggests that tenascin may be readily catabolized by extracellular matrix-degrading proteinases found in the pathophysiological conditions.

The mouse chimera during intrauterine stages: immunohistochemical analysis with the C3H strain-specific antibody.

Our objective was to establish an immunohistological method for analysis of chimerism in mouse chimeras at embryonic stages with an anti-C3H strain-specific antigen (CSA) antibody. We developed an effective new method to retain CSA antigenicity with good morphology of embryonic tissues by using microwave irradiation (MWI) for pre-fixation, 95% ethanol/1% acetic acid as post-fixative solution, and polyester wax as embedding material. We used a biotinylated mouse monoclonal anti-CSA antibody, peroxidase-avidin, and silver amplification. These procedures were successful in demonstrating the chimerisms in various tissues of C3H<-->Balb/c chimeras at different embryonic stages and postnatal days. In chimeras at Days 7 and 7.5 post coitum (p.c.), both genotypes were clearly identified and well intermingling in every embryonic tissue (embryonic ectoderm, mesoderm, extra-embryonic ectoderm, ectoplacental cone, amnion, and chorion). Chimerisms at Day 14.5 p.c. were also clearly observed in mesencephalon, neural retina, spinal cord, lung, kidney, and liver. We concluded that the present immunohistological procedures for analysis of chimerism during embryonic periods will give us insightful information about dynamic histological changes such as cell proliferation, migration, selection, and death during organogenesis.

Tenascin in breast cancer development--is epithelial tenascin a marker for poor prognosis?

(1) In mouse mammary gland development, immunoreactive tenascin (TN) is expressed in the dense mesenchyme surrounding the epithelial component of 14-day embryos, endbuds at puberty, and tumors. (2) Cells that produce TN are myofibroblastic and are characterized by nuclear invaginations, rough endoplasmic reticulum, and pinocytotic vesicles. These cells are not normally present in the stroma of mammary glands but present in cancer stroma, originating probably from fibroblasts differentiated under the influence of TGF-beta 1 stimulation. (3) Breast cancer cells are capable of synthesing TN under certain conditions. TN-non-producing MCF7 cells can produce TN when co-cultured with embryonic fibroblasts or with their conditioned medium. (4) Nine primary human breast cancers were examined for TN expression by in situ hybridization. TN mRNA was expressed in all nine cases in the stroma and in four cases in carcinoma cells as well. (5) Immunohistochemistry for TN was performed in human breast cancers, and it was found that the five-year survival after surgery was markedly lower in the group whose cancer cells were positive [corrected] for TN. TN expression in cancer cells appears to indicate poor prognosis.

Clonal analysis of glandular stomach carcinogenesis in C3H/HeN<==>BALB/c chimeric mice treated with N-methyl-N-nitrosourea.

The clonal growth of gastric carcinomas was investigated immunohistochemically in C3H<==>BALB/c chimeras using a strain specific antibody. C3H, BALB/c and chimeric mice were given N-methyl-N-nitrosourea 0.5 mg/mice once a week for a total of 10 times by intragastric intubation and observed until week 50. In normal gastric mucosa of the chimeras, each gland was composed entirely of C3H strain specific antigen (CSA)-positive or -negative cells and no mixed glands were found. Cells of all adenomatous hyperplasias and adenocarcinomas in chimeric mice were, in each case, homogeneous for one or other of the parental types, while comprising both surface mucous cell and pyloric gland cell forms. The results clearly suggest that individual cancers are derived from single cells with multi-potential activities and that cellular differentiation of gastric cancer cells occurs secondarily.

Overexpression of caltractin gene in tumor-infiltrating lymphocytes.

Differential display is a technique which relies on the polymerase chain reaction to identify messenger RNA differences between related tissue samples. We have employed an improved differential display technique, called fluorescent differential display (FDD), to identify the genes that are differentially expressed in normal and malignant mammary tissues. From FDD fingerprints, we identified changes in intensity of approximately 3% (185 bands) of a total of 5, 837 bands. Each of these 185 bands represented a differentially expressed gene, and we focused our attention on the expression of the gene for caltractin, a member of the calcium-binding EF-hand protein superfamily. Northern blot analysis revealed that the level of mRNA for caltractin was higher in breast carcinoma than in corresponding normal tissue in all cases tested (5/5). Moreover, high-level expression of the gene for caltractin was also recognized in other malignant tumors, such as hepatocellular carcinoma (HCC), gastric cancer and leiomyosarcoma. The results of in situ hybridization showed strong stainings for caltractin mRNA in tumor-infiltrating lymphocytes (TIL), but not in malignant tumor cells. Our data suggests that the caltractin gene might be associated with the function of TIL.