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.

Clonal behavior of a rat liver cell line and its modification by repeated treatments with a carcinogenic polycyclic hydrocarbon.

A rat liver cell line, Lew A1, was isolated from W/LEW rats. It had the normal female karyotype in the lower passage numbers, but in the higher passages it was aneuploid. This line was passaged 65 times, produced rat serum albumin, and consisted of an apparently homogeneous population of typical epithelial cells. The cells also had high levels of the inducible aryl hydrocarbon hydroxylase enzyme complex. A series of experiments described here defined the normal clonal behavior of this line and its modification by repeated treatments with a carcinogenic polycyclic hydrocarbon, 7,12-dimethylbenz[a]anthracene. The results were discussed with particular reference to metastasis, preneoplastic changes, and neoplastic progression.

Spontaneous cell loss during growth of postconfluent primary cultures from mammary adenocarcinomas.

Growth properties of cells cultured from primary mammary tumors of C3H mice have been analyzed. Cells were seeded at 2 different densities (1 X 10(5) and 5 X 10(5)/sq cm) and were supported with a culture fluid containing 10% fetal calf serum and 5 mug insulin per ml. Mitosis continued after confluence was achieved, but cells did not accumulated in the monolayer; rather, certain cells were released into the culture fluid. Very few cells detached in this way from subconfluent cultures. Relased cells multiplied vigorously if replated. The release of these cells was strongly depressed by adrenal steroids, but other manipulations of culture conditions (hormones, culture substratum) influenced the release process much less. Analyses of release kinetics and observations of detachment with the scanning electron microscope suggested that tumor cells that became spheroid (including mitotic cells), and hence partly detached from the culture dish, were unable to reflatten into the monolayer because neighboring nonmitotic cells had spread onto the vacated culture surface. Eventually, such rounded cells apparently lost altogether their attachment to the culture dish. The release process may be related to the "critical phase" transition and to the sarcomatous transformation observed in long-term cultures from mouse epithelial tumors. The event could also reflect the tendency in vivo for cells of mammary tumors to slough into the lymphatics and blood vessels.

Invasion of endothelial cell monolayers on collagen gels by cells from mammary tumor spheroids.

Suspensions of multicellular mammary tumor spheroids (MTS) were allowed to interact with confluent monolayers of endothelial cells cultured on top of collagen gels. A number of early and late interactions between MTS and endothelial cell monolayers occurred. The early phase was characterized by the attachment of MTS to the culture and retraction of endothelial cells near the attached spheroid. Only these early interactions were observed up to 8 hr after addition of the MTS. Thereafter, cells from MTS migrated away from the spheroids. The late phase was characterized by cells of the MTS spreading on top of the collagen gel, moving underneath the edges of the endothelial cells, extending as cords of cells on top of the endothelium, and invading into the collagen matrix. During both the early and late phases, cells from the MTS were distinguished from the endothelial cells by the intense staining of tumor cells with Giemsa and the presence of microvilli found only on tumor cells. Attached MTS, which were noted at 2 hr after addition (the earliest time examined), increased in number for up to 12 hr. Polyionic compounds known to affect cell surface charge were found to reduce the numbers of attached MTS. The results demonstrate that the system described in this study can provide a useful model for analyzing the mechanisms of tumor embolus interaction with blood vessel walls.

Glycosaminoglycan synthesis by a cell line (C1-S1) established from a preneoplastic mouse mammary outgrowth.

We have measured the synthesis of several types of glycosaminoglycans by a line of mouse mammary epithelial cells (C1-S1) established from a hyperplastic nodule outgrowth. These epithelioid cells do not grow readily in vivo. Subconfluent monolayer cultures of C1-S1 cells produced more hyaluronic acid than heparan sulfate, but the opposite was true in confluent cultures. At saturation density in culture, the cell surface glycosaminoglycan of C1-S1 cells was approximately 80% heparan sulfate. For comparison, data are also reported on two related tumorigenic sublines (+SA and -SA) established from a spontaneous tumor in a hyperplastic outgrowth. These cells produced mostly hyaluronic acid even when confluent. Furthermore, the net rate of hyaluronic acid synthesis was higher in the more aggressive tumor cells (+SA). The data are consistent with the interpretation that a hyaluronic acid-rich, heparan sulfate-poor environment is associated with the growth of mammary epithelial cells and conversely that a heparan sulfate-rich environment may restrict growth. The glycosaminoglycan environment may thus contribute to growth modulation in vivo.

Elevated high mobility group-I(Y) gene expression is associated with progressive transformation of mouse mammary epithelial cells.

The high mobility group (HMG) proteins I and Y are well characterized nonhistone chromosomal proteins which bind to A.T-rich regions of DNA, and may regulate gene expression and/or DNA replication. We utilized a series of mouse mammary epithelial preneoplastic and tumor cell lines to explore the relationship between neoplastic transformation and HMG-I(Y) gene expression. The cell lines used in this study were originally derived from a single hyperplastic outgrowth, and exhibit a distinct gradient of preneoplastic to highly metastatic transformation states. We measured the levels of HMG-I(Y) gene expression in these cell lines during the different phases of cell growth in culture. At both subconfluent and confluent cell densities, elevated levels of HMG-I(Y) mRNA were directly correlated with the relative degree of neoplastic transformation and metastatic progression of these cells. HMG-I(Y) mRNA levels were always highest in proliferating cells. However, the differences in HMG-I(Y) gene expression between the cell lines were greatest at confluent cell density, when the cells were not actively proliferating. HMG-I(Y) mRNA was detectable in normal primary mouse mammary epithelium proliferating in culture. However, the amount was much less than that measured in the cell lines, indicating that elevated HMG-I(Y) gene expression was also directly correlated with the conversion of normal mammary epithelium to the preneoplastic immortalized state. Southern blot analysis showed that alterations in HMG-I(Y) loci are also associated with the preneoplastic to neoplastic conversion of these cell lines, and this change may involve a gene conversion event between two different HMG-I(Y) loci. These results indicate that there is a strong correlation between elevated HMG-I(Y) gene expression and the progressive transformation of mouse mammary epithelial cells.

Interaction of metastatic tumor cells with bovine lens capsule basement membrane.

The use of bovine lens capsule basement membrane as a model substratum for studies of invasion and extravasation by metastatic tumor cells is described. The abilities of three independently isolated pairs of metastatic variant cell lines to digest the purified substrates, laminin, type IV collagen, and type I collagen, were compared with their abilities to solubilize isotope from 125I-labeled lens capsule basement membrane matrix. The cell lines used were +SA and -SA mouse mammary adenocarcinoma cells, RT7-4bs and RT7-4b-Ls rat hepatocarcinoma cells, and B16-F1 and B16-F10 mouse melanoma cells. In general, imperfect correlations of lytic activity with metastatic ability were found for the purified substrate digestions, but, for each pair of variants, the more metastatic tumor cell line was always able to solubilize more surface-bound isotope from the lens capsule. Visual evidence of tumor cell-associated digestion of lens capsule basement membrane was obtained using transmission electron microscopy. Mouse mammary carcinoma cells attached more rapidly to lens capsule than to endothelial cell monolayers or tissue culture plastic. We next added endothelial cells to the model substrate. Aortic endothelial cells grew well on lens capsules without apparent synthesis of additional basement membrane matrix. In additional studies, the lens capsule was used in a chamber apparatus to demonstrate that cellular invasion of the full thickness of this basement membrane structure could be demonstrated and readily quantitated. Our results indicate that bovine lens capsule is a particularly versatile basement membrane structure useful for studies of tumor cell invasion and extravasation. In addition, the comparison of purified substrate digestions with lens capsule matrix digestion indicates the desirability of also using a matrix digest when correlating lytic abilities of tumor cells with their metastatic abilities.

Glycosaminoglycan synthesis by subpopulations of epithelial cells from a mammary adenocarcinoma.

Glycosaminoglycan synthesis by two subpopulations of a mouse mammary tumor cell line was compared. The two sublines express distinctly different growth characteristics in vitro and in vivo which indicate differences in growth regulation. Newly made glycosaminoglycans were recovered from the culture media, the cell surfaces, and residual cellular material. The cell population which grows more aggressively in vivo (+SA subline, a subline that grows in soft agarose) incorporated about 8 times more [14C]glucosamine per cell into total glycosaminoglycans than did the slower-growing population (-SA subline, which does not grow in soft agarose). Appropriate control experiments indicated that the apparent difference in rates of synthesis was not due to discrepancies in glucosamine uptake. The main residual cellular molecule labeled was heparan sulfate, but the predominant molecule at the cell surface and in the culture fluid was hyaluronic acid. Overall, +SA cells synthesized more hyaluronic acid and -SA cells synthesized more heparan sulfate; in both cell populations, these two molecules accounted for about 90% of total glycosaminoglycans produced.

In vitro replication potential of serially passaged mammary parenchyma from mice with different reproductive histories.

Growth properties of multicellular units (organoids) of mouse mammary parenchyma have been analyzed. These intact units grew differently in collagen-matrix cultures than did dispersed cells prepared from them. The latter actively migrated in the collagen matrix and reorganized themselves into multicellular structures before producing three-dimensional protuberances in gel. Terminal unit (end-bud/alveoli)-enriched fractions grew more extensively than did ducts, as predicted from growth patterns in vivo. To assess the growth potential and the relationships between replication history in vivo and replication potential in vitro in mammary parenchyma, intact terminal units from mammary glands of mice of different ages and with different reproductive histories were isolated and their growth characteristics compared. Terminal-unit organoids were cultured in collagen gel matrix and passaged weekly for up to 5 weeks. Morphology, growth rates, and growth fractions were compared among organoids from young virgin, old virgin, monoparous, and multiparous mice. Morphologies observed in various passages of organoids from the groups of mice were similar. Organoids from old virgin and multiparous mice declined in growth rate for four passages and then growth rate increased again during the fifth passage. (However, fifth-passage organoids failed to form tumors if implanted in syngeneic mice in vivo.) Growth of organoids from either old or young virgin mice was less at any given passage than tissue from multiparous mice of similar age. Growth fractions of organoids from old parous mice were the same as those from old virgin mice but reached the same maximum fraction faster. Later passage organoids from the different mouse groups responded morphologically to the hormone combination of estrogen, progesterone, prolactin, and cortisone but not respond to cholera toxin. These results suggest that an animal's hormonal history (altered profoundly by pregnancy and lactation) may be as important as chronological age in determining subsequent growth potentials of mammary epithelium.

Hydrocortisone inhibits the spontaneous detachment of viable mammary tumor cells from monolayer cultures.

Primary mammary adenocarcinomas of C3H mice were dissociated to single cells. These cells were seeded at a density of 5 x 10(5)/cm2 substrate area and were grown as monolayers for 36 days in high-serum medium supplemented with insulin. Cultures became confluent within a few days and confluence was maintained throughout the culture period. Viable cells (representing amitotic cell population) were released spontaneously from the confluent monolayer and could be harvested from the culture fluid. The rate of cell release declined after about 30 days, apparently due to mitotic pool depletion. Release of cells from the monolayer was strongly depressed in medium containing hydrocortisone. The cell release event and its alteration by hydrocortisone can be interpreted as reflecting the propensity towards shedding of cells from these tumors in vivo and the alteration of metastatic incidence by adrenal steroids.

Milk-fat synthesis by lobules prepared from rabbit mammary gland: response to insulin, corticosterone, prolactin and progesterone.

Multi-alveolar mammary structures (mammary lobules) were prepared from mammary glands of pseudopregnant rabbits by controlled digestion with collagenase and hyaluronidase. The overall rate of fatty acid synthesis and the proportion of milk-specific fatty acids (C8:0 and C10:0) synthesized by these lobules when cultured with insulin, corticosterone and prolactin were measured. Maximum response to physiological concentrations of prolactin (1.1 or 2.2 nmol/l) occurred in the presence of insulin (1.7 mumol/l) and corticosterone (0.58 mumol/l). In general, the results obtained on the effect of progesterone were negative. Though explants showed a ninefold greater response to prolactin per mg DNA than did mammary lobules, the latter have the advantage of being easily prepared for culture in large numbers. Reduction to below 500 microns diameter and culture in conditions which allow cell outgrowth onto plastic limited their response to prolactin. The probable roles of membrane damage by digesting enzymes and of tissue architecture in limiting prolactin response are discussed.

CaO--P2O5--Na2O-based sintering additives for hydroxyapatite (HAp) ceramics.

We have assessed the effect of CaO--P2O5--Na2O-based sintering additives on mechanical and biological properties of hydroxyapatite (HAp) ceramics. Five different compositions of sintering additives were selected and prepared by mixing of CaO, P2O5, and Na2CO3 powders. 2.5 wt% of each additive was combined with commercial HAp powder, separately, followed by ball milling, and sintering at 1250 degrees C and 1300 degrees C in a muffle furnace. Green and sintered densities of the compacts were analyzed for the influence of additives on densification of HAp. Phase analyses were carried out using an X-ray diffractometer. Vickers microhardness testing was used to evaluate hardness of sintered compacts of different compositions. A maximum microhardness of 4.6 (+/- 0.28) GPa was attained for a composition with 2.5 wt% addition of CaO:P2O5:Na2O in the ratio of 3:3:4. Results from mechanical property evaluation showed that some of these sintering additives improved failure strength of HAp under compressive loading. Maximum compressive strength was observed for samples with 2.5 wt% addition of CaO. Average failure strength for this set of samples was calculated to be 220 (+/- 50) MPa. Cytotoxicity, and cell attachment studies were carried out using a modified human osteoblast cell line called OPC-1. In vitro results showed that these compositions were non-toxic. Some sintering aids enhanced cell attachment and proliferation, which was revealed from SEM examination of the scaffolds seeded with OPC-1 cells.

Identification and characterization of a novel angiotensin binding site in cultured vascular smooth muscle cells that is specific for the hexapeptide (3-8) fragment of angiotensin II, angiotensin IV.

This study demonstrates the existence of a previously unrecognized class of angiotensin binding sites on vascular smooth muscle that exhibit high affinity and specificity for the hexapeptide (3-8) fragment of angiotensin II (AngIV). Binding of [125I]AngIV is saturable, reversible and describes a pharmacologic profile that is distinct and separate from the classic AT1 or AT2 angiotensin receptors. Saturation binding studies utilizing cultured vascular smooth muscle cells obtained from bovine aorta (BVSM) revealed that [125I]AngIV bound to a single high affinity site with an associated Hill coefficient of 0.99 +/- 0.003, exhibiting a KD = 1.85 +/- 0.45 nM and a corresponding Bmax = 960 +/- 100 fmol mg-1 protein. Competition binding curves in BVSM demonstrated the following rank order effectiveness: AngIV > AngII(3-7) >> AngIII > Sar1,Ile8 AngII > AngII > AngII(1-7) > AngII(4-8), DuP 753, PD123177. The presence of the non-hydrolyzable GTP analog GTP gamma S, had no effect on [125I]AngIV binding affinity in BVSM. The presence of this novel angiotensin binding site on smooth muscle in high concentration suggests the possibility that this system may play an important, yet unrecognized role in vascular control.

Microprocedure for in situ nick translation of chromosomes.

We have modified the procedure of in situ nick translation to shorten the autoradiographic exposure time from 1 month to 3 days and reduce the volume of nick translation solution by a factor of at least 10. The modified procedure can be carried out on individually chosen chromosome spreads. The procedure was used on chromosome spreads of three related lines of mouse mammary epithelium (+SA, -SA, CL-S1) with different degrees of tumorigenicity. We found that the autoradiographic silver grains that are observed following in situ nick translation were often placed at the apparent junction site of chromosome translocations or at the breakpoint of chromosomal pieces. We found also that silver grains were located above double minute chromosomes, which suggests that there are active genes in double minutes.

Immunosuppressant inhibition of P-glycoprotein function is independent of drug-induced suppression of peptide-prolyl isomerase and calcineurin activity.

PURPOSE: P-glycoprotein is a 170-kDa plasma membrane multidrug transporter that actively exports cytotoxic substances from cells. Overexpression of P-glycoprotein by tumor cells is associated with a multidrug-resistant phenotype. Immunosuppressive agents such as cyclosporins and macrolides, have been shown to attenuate P-glycoprotein activity. However, the mechanism by which some immunosuppressants inhibit P-glycoprotein function has not been determined. Since cyclosporin and macrolide immunosuppressants inhibit calcineurin (CaN) phosphatase and FKBP12 peptideprolyl isomerase (FKBP12 PPI) activity, studies were conducted to determine if these effects are directly related to the inhibitory effects these immunosuppressants have on P-glycoprotein function. METHODS: Western blot analysis was performed to assess CaN and FKBP12 protein levels in P-glycoprotein-negative (MCF-7) and -positive (MCF-7/Adr) breast cancer cell lines. P-glycoprotein function was determined by intracellular doxorubicin accumulation and/or cytotoxicity assays before and after CaN and FKBP12 were independently inhibited by pharmacological antagonists. RESULTS: CaN and FKBP12 levels were similar in MCF-7 and MCF-7/Adr cells. P-glycoprotein function was not affected by treatment of P-glycoprotein-expressing MCF-7/Adr cells with CaN and FKBP12 antagonists. CONCLUSIONS: These results demonstrate that the inhibitory effects of immunosuppressive agents on P-glycoprotein function are independent of CaN or FKBP12 PPI activity.

Effects of dietary conjugated linoleic acid on lymphocyte function and growth of mammary tumors in mice.

We studied the effects of conjugated linoleic acid (CLA) on lymphocyte function and growth of a transplantable murine mammary tumor. In experiment 1, eight-wk-old female Balb/c mice (n = 8/group) were fed 0.1%, 0.3% or 0.9% CLA for 3 or 6 wk. Lymphocyte proliferation, interleukin-2 production and lymphocyte cytotoxicity were assessed using splenic lymphocytes. Plasma CLA concentrations increased in a dose-dependent manner with CLA feeding. Lymphocyte proliferation in mice fed 0.3% and 0.9% CLA was enhanced in phytohemagglutinin-induced but not in concanavalin A- or lipopolysaccharide-stimulated cultures. Production of IL-2 also was stimulated by CLA. In contrast, CLA had no effect on lymphocyte cytotoxicity. In experiment 2, mice (n = 20/treatment) were fed the same diets for 2 wk before being infused with 1 x 10(6) WAZ-2T metastatic mammary tumor cells into the right inguinal mammary gland. Tumor volume and latency were recorded for 45 d. Dietary CLA did not affect mammary tumor growth. Tumor latency, tumor incidence and tumor lipid peroxidation activity also were unaffected by CLA. Body weight and feed intake were similar among treatments. Therefore, dietary CLA modulated certain aspects of the immune defense but had no obvious effect on the growth of an established, aggressive mammary tumor.

Effects of MgO-CaO-P2O5-Na2O-based additives on mechanical and biological properties of hydroxyapatite.

In this research, we improved densification, hardness, and compression strength of synthetic hydroxyapatite (HAp) ceramics by introducing small quantities of MgO-CaO-P(2)O(5)-Na(2)O-based sintering additives. Biological properties of HAp were not altered by this procedure. Phase analyses were performed by using a Philips Xpert fully automated diffractometer with Co K-alpha radiation to understand the influence of additives on phase purity in the final products. All compositions were characterized at green and sintered densities to understand the influence of additives on densification. Some of the compositions showed >40% increase in Vickers microhardness compared with pure HAp processed under the same conditions. Improvement in compression strength was also detected in some compositions. In vitro biological testing used a modified human osteoblast cell line to test biocompatibility, cell attachment, and cell proliferation. All these compositions were nontoxic and biocompatible. Our results indicate that MgO-CaO-P(2)O(5)-Na(2)O-based sintering additives can be used to improve both mechanical and biological properties of HAp ceramics.

Growth of cells in culture treated with the soluble component of volcanic ash from Mount St. Helens.

Volcanic ash was collected immediately after the eruption of Mount St. Helens on May 18, 1980. This ash was extracted with water. The elemental composition of the extracted portion was determined by atomic absorption spectrometry. The aqueous extract was applied at high concentrations (up to 37.5 micrograms/ml) to non-confluent mixed cultures of mouse lung cells. Even after treatment for up to 10 days, cell number was typically unaffected by the ash extract. Cell viability was also unaltered, and no grossly observable changes were noted in the cells by light microscopy. We conclude that the water-soluble portion of the ash we tested does not markedly affect growth of the cells most at risk, those of the lung.