Effects of defined medium,fetal bovine serum,and human serum on growth and chemosensitivities of human breast cancer cells in primary culture: Inference for in vitro assays

Summary We compared the effects of defined medium, fetal bovine serum (FBS) and human serum (HuS) on the growth and responses to chemotherapeutic agents of human breast cancer cells in primary culture. Normal and tumor tissues were dissociated to small aggregates and single cells and seeded onto collagen-gel-coated wells in defined medium or medium supplemented with 5% FBS or 5% HuS. In all cases examined, defined medium and medium containing HuS were superior to medium containing FBS in supporting growth of both normal and tumor cell cultures. However, cultures in defined medium showed an initial cell loss. Cells from the same tumor cultured in different media varied in their responses to chemotherapeutic agents. In light of these results, medium supplemented with HuS, which promoted attachment of these cells in culture and stimulated their growth, should be the most appropriate nutrient environment for determining the effects of therapeutic agents on cells as it most closely resembles the in vivo situation. Because there were also variations in growth rates and chemosensitivities of tumor cells cultured in different human serum samples, we suggest that optimal conditions in which to culture these cells include the serum of the patient whose tumor is removed. This serum may provide host factors that influence cell growth and interact with exogenous factors. This work was supported by a grant from the National Cancer Institute of Canada and funds contributed by Mr. B. T. Wharton in memory of his wife, Nadia. J. T. Emerman is a research scholar of the National Cancer Institute of Canada.     

Behavior of primary and serially transplanted estrogen-dependent renal carcinoma cells in monolayer and in collagen gel culture

Summary When estrogen is present in culture medium, enzyme-dissociated cells from estrogen-induced primary renal tumors in Syrian hamsters and from 1st to 4th serially transplanted carcinomas in monolayer culture contain progesterone receptor levels that are similar and comparable to those in tumors in vivo (i.e., 2 pmol/3×10⁶ cells). Despite the similarity of receptor levels in cultured cells isolated from primary and transplanted tumors, the ability of cells to be maintained in culture differs considerably from one tumor stage to another. When cultured as monolayers in plastic flasks, isolated cells from primary tumors exhibit a marked decline in cell number after 4 to 6 d in culture. On the other hand, monolayer-cultured cells from first and second transplantation tumors remain essentially constant in cell number over a 2 wk culture period and cells from third transplantation tumors undergo a two- to threefold increase in cell number during 2 wk in culture. When primary tumor cells are cultured in collagen gels, the decline in cell number over a 2 wk culture period is prevented and progesterone receptor levels remain elevated. Cells cultured from first transplantation tumors exhibit a delayed decline in cell number beginning after 2 wk in monolayer culture. The decline in cell number in monolayer culture, like that for cells from primary tumors, can be prevented by culturing cells from first transplantation tumors in collagen gels. Neither cells from primary nor first transplantation tumors exhibit significant increases in cell number in collagen gels. Increasing the serum concentration of growth medium to 30% does not stimulate growth of cells under these conditions. Cells isolated from fourth transplantation tumors undergo a fourfold increase in cell number over a 1 month culture period whether cells are cultured as monolayers or in collagen gels. This investigation was supported by Grant CA 22008 awarded by the National Cancer Institute, Department of Health, Education and Welfare, and by institutional research funds from Marquette University.     

In vitro characterization of estrogen induced syrian hamster renal tumors: Comparison with an immortalized cell line derived from diethylstilbestrol-treated adult hamster kidney

Summary Primary diethylstilbestrol-induced kidney tumors from Syrian hamsters were grown in vitro and maintained in culture for 6 mo. Combined immunohistochemical studies using antibodies to intermediate filaments and ultrastructural studies of tumor cells in culture exhibited characteristics similar to tumor cells in vivo. Furthermore, the cells manifested transformed properties in culture; they grew both as multilayered colonies attached to the tissue culture substrate and as floating multicellular colonies (spheroids). When cultured cells were injected into diethylstilbestrol-treated recipient hamsters, tumors developed at the injection sites. In contrast, renal tubules or whole kidney cortex from control hamsters cultured in the same medium underwent only short-term growth, with senescence developing after approximately 1 mo. However, cell cultures of kidney cortex from animals treated in vivo for 5 mo. with diethylstilbestrol formed a cell line. This diethylstilbestrol-induced cell line has been maintained in culture for 1.5 yr and has the following characteristics: a) it is anchorage-dependent, b) it is negative in in vivo tumorigenicity tests, and c) cultured cells are histochemically and ultrastructurally similar to cultured tumor cells. This culture system should prove to be of use in studying hormonal carcinogenesis in vitro. This study was supported by the Medical Research Service, Department of Veterans Affairs, Washington, DC, and by grant CA-22008 from the National Cancer Institute, NIH, DHHS, Bethesda, MD.     

Routine culturing of normal,dysplastic and malignant human mammary epithelial cells from small tissue samples

Summary We compared the growth and morphology of normal, dysplastic and malignant human mammary epithelial cells (HMEC) in medium containing 5% human serum, a serum-free medium (32) and serum-free medium with a low Ca⁺⁺ concentration. Tissues were dissociated and epithelial organoids or single cells were seeded onto collagen-coated dishes. The cells grew in serum-containing medium, but growth of fibroblasts was also stimulated. The serum-free medium consistently selected for and stimulated the growth of epithelial cells. There was little advantage in reducing the Ca⁺⁺ concentration to further increase cell yield. This serum-free primary culture system allows us to routinely prouce sufficient numbers of HMEC from small tissue samples for molecular biological investigations. Furthermore, the maintenance of cells in a defined medium can provide a system for evaluating the direct effects of factors on gene expression. This work was supported by a grant from the National Cancer Institute of Canada and funds contributed by Mr. B. T. Wharton in memory of his wife, Nadia.     

Morphogenic activity of fibroblast growth factor-2 on primary neural precursor cells in three-dimensional culture

Mouse neural precursor cells (NPC) were dissociated from fetal heads at the 10th day of gestation. When clumps of NPC were cultured in collagen gel, they grew and reorganized neural tube-like structures in medium containing fetal calf serum at 10% and supplemented with insulin, transferrin, cholera toxin and selenite. However, dissociated NPC died when they were cultured in collagen gel at low density in the same medium. Addition of fibroblast growth factor-2 (FGF-2) to this culture stimulated growth of NPC and formation of neural tube-like structures. The requirement for FGF-2 disappeared in high seeding density culture: they grew and formed neural tube-like structures without FGF-2. The structures formed in collagen gel were immunohistochemically positive against anti-FGF-2 antibody. The results show that the three-dimensional culture system provides a useful tool to study the roles of FGF-2 in morphogenesis of the central nervous system.     

Release of acetylcholinesterase by cultured spinal cord cells

The release of acetylcholinesterase from neurons was studied using cultured chick-embryo spinal-cord cells. Cells dissociated from 12-day-old chick-embryo spinal cords were grown in culture for 10-12 days. Numerous well differentiated spinal neurons were found after 7-10 days in culture. Acetylcholinesterase activity per dish increased by 60-fold from days 2-12. Acetylcholinesterase was released into the surrounding media by the cells when they were incubated either in the standard culture medium or the serum-free medium. Acetylcholinesterase release was significantly reduced when protein synthesis and microtubules were disrupted by cycloheximide and colchicine, respectively. Histochemical localization of acetylcholinesterase indicated that the synthesis and relase of acetylcholinesterase are attributable to neurons. Cultured chick-embryo brain and neuroblastoma cells also released acetylcholinesterase into the media. These results are discussed with regard to possible physiological roles for acetylcholinesterase secretion from neurons.     

Growth and characterization of multicellular tumor spheroids of human bladder carcinoma origin

Summary We have examined the MGH-U1 human bladder carcinoma cell line and 12 primary bladder carcinoma biopsies for their ability to form spheroids in suspension culture and in multiwell dishes. MGH-U1 cells formed tightly packed spheroids with a necrotic center and viable rim whereas three sublines formed loose aggregates only. Spheroids formed from as few as 100 MGU-U1 cells placed into multiwells. MGH-U1 cells derived from spheroids formed new spheroids more rapidly and consistently than cells derived from monolayer culture. Spheroid diameter increased at a rapid rate of ∼100 μm/d in multiwell dishes, and necrosis occurred only in spheroids of diameter >1 mm. Spheroids placed in spinner culture at a higher concentration (∼1.5 spheroids/ml) grew more slowly and developed necrosis at smaller diameters. The width of the viable rim of spheroids grown in spinner culture was maintained at ∼190 μm over a wide range of spheroid diameters (400 to 1000 μm). Sequential trypsinization of spheroids, which stripped layers of cells from the spheroids, demonstrated no difference in the plating efficiency of cells derived from varying depths into the spheroid. Only one of the 12 primary bladder biopsy specimens demonstrated an ability to form spheroids. This biopsy, designated HB-10, formed spheroids that grew linearly over 40 d, formed colonies in methylcellulose culture and grew as xenografts in immune-deprived mice. These studies characterize the MGH-U1 spheroids that are useful in vitro models to study the effects of various treatments for solid tumors and demonstrate the limited capacity of cells from primary human bladder biopsies to form spheroids. Supported in part by a grant from the National Cancer Institute of Canada and by grant CA29526 NCI through the National Bladder Cancer Project, U.S.A.     

Analysis of rat adrenal cells in primary culture separated by velocity sedimentation

Summary Cell separation was used to follow the fate of the cortical cells of the adrenal gland in primary culture, and to assess some of the changes that occur as cells adapt to culture conditions. Primary cultures of rat adrenal gland were dissociated with trypsin and separated by velocity sedimentation at unit gravity. After two days in culture, cells showed a reproducible sedimentation profile consisting of two classes of cells with mean sedimentation rates of 5.8 and 2.1 mm/h, and a third sedimentation peak consisting mainly of nuclei at 0.5mm/h. All populations continued to incorporate ³H-thymidine in relatively constant proportion throughout the culture period, but the relative number of cells in the 2.1 mm/h peak increased two-fold in the last few days of primary culture. Cells labelled in primary culture, but separated after an additional 5 days in secondary culture had lost proportionately more labelled cells from the 5.8 mm peak. The results suggest that cells of the 2.1 mm peak survive longer in culture in a post-replicative condition.Work reported in this paper was performed while the author was a Research Fellow of the National Cancer Institute of Canada in the laboratory of Dr. N. Auersperg, Cancer Research Centre, University of British Columbia, Vancouver, B.C., Canada     

The influence of cell shape on the induction of functional differentiation in mouse mammary cells in vitro

Summary To define more clearly the in vitro conditions permissive for hormonal induction of functional differentiation, we cultured dissociated normal mammary cells from prelactating mice in or on a variety of substrates. Cultivation of an enriched epithelial cell population in association with living adult mammary stroma in the presence of lactogenic hormones resulted in both morphological and biochemical differentiation. This differentiation, however, was not enhanced over that seen when the cells were associated with killed stroma, provided that the killed stroma had a flexibility similar to that of the living stroma. Cells cultured in inflexible killed stroma usually did not differentiate. Cells cultured within the flexible environment of a collagen gel, but removed from the gas-medium interface, differentiated in a manner similar to those cultured in flexible stroma. Cells cultured on the surface of an attached collagen gel were squamous, and their basolateral surfaces were sequestered from the medium; they did not differentiate. Cells cultured on floating collagen gels were cuboidal-columnar, with basolateral surfaces exposed to the medium, and showed good functional differentiation. Cells cultured on inflexible floating collagen gels were extremely flattened and had exposed basolateral surfaces, and showed no evidence of functional differentiation. We infer that assumption of cuboidal to columnar shapes similar to those of mammary cells in vivo may be important to the induction of functional differentiation in vitro. The additional requirement of basolateral cell surface exposure also is important. This work was supported by U.S. Public Health Service Grants CA-05045 and CA-09041 from the National Cancer Institute, Bethesda, MD.     

The androgen receptor does not mediate progestin regulation of progesterone biosynthesis in cultured rat granulosa cells

In the present investigation the influence of androgens and progestins on the FSH modulation of progesterone biosynthesis was studied in cultured rat granulosa cells. Cells obtained from the ovaries of immature estrogen treated rats were cultured for three days in serum free medium or in medium supplemented with FSH or CPA, with or without reduced androgen DHT or the synthetic progestin R5020 alone or in combination with the anti-androgen CPA. Treatment with FSH increased pregnenolone, progesterone and 20 alpha-OHP accumulation in the culture medium 20-, 14- and 7-fold, respectively. Furthermore FSH increased the activity of the enzyme 3 beta-HSD. Concurrent treatment with DHT or R5020 augmented the FSH stimulated steroidogenesis of cultured cells. The androgen enhancement of FSH stimulated steroidogenesis of cultured granulosa cells was blocked by concomitant treatment with CPA, whereas treatment of cultures with anti-androgen did not affect the stimulatory effect of the synthetic progestin R5020.     

Dose response of insects to malignant transformed cells

Summary Graded doses of cell suspensions of an embryonic cockroach cell line in its 290th passage were injected into the hemoceles of hosts of the homologous species,Blattella germanica, of different ages. All insects injected with viable cells suffered paralysis, progressive loss of appendages, and premature death. The time of death depended on the number of cells inoculated. Histological studies of the afflicted insects showed that the injected cells were metastatic and invasive, producing tumors, especially in the fat body. Tumors were transplantable and malignant in secondary hosts. Tumors were not produced by cell-free spent medium, or homogenized or sonicated cell breis. The cause of paralysis and loss of appendages was not known, although large aggregations of the injected cells could be seen in the leg musculature in the thorax. Cells were recovered from the tumors and grown again in culture. The line was predominantly diploid (62%) with some heteroploid cells (38%). This work was supported in part by Research Grant AI 09914 from the National Institutes of Health. This is Paper 10,949, Scientific Journal Series, Minnesota Agricultural Experiment Station.     

Effect of polypeptide hormones on stimulation of casein secretion by mouse mammary epithelial cells grown on floating collagen gels

Summary The in vitro effects of protein hormones on the stimulation of casein secretion by mouse mammary epithelial cells were studied. Mouse mammary glands were enzymatically dissociated and used immediately or were stored frozen and thawed just before use. Cells were cultured on floating collagen gels in the presence of insulin, cortisol and a pituitary or placental polypeptide hormone. Casein, released into the medium, was assayed by a radioimmunoassay against one of the components of mouse casein. Mammary cells released casein into the medium in the presence of as little as 10 ng of ovine prolactin per ml of medium. Human growth hormone stimulated the casein secretion to the same extent as prolactin. Human placental lactogen, ovine and bovine growth hormones were less stimulatory. Luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone had no effect on the stimulation of casein secretion. This investigation was supported by Grant No. CA 05388 awarded by the National Cancer Institute, DHEW, and by Cancer Research Funds of the University of California.     

Duct,exocrine, and endocrine components of cultured fetal mouse pancreas

Summary Twenty to twenty-two days postcoitum mouse fetal pancreas organ bits were cultured on the dermal surface of irradiated pigskin as a substrate. The medium used for long term culture consisted of Eagle’s Minimum Essential Medium with the addition of 10% bovine serum, 0.02 U/ml insulin, 0.025 μg/ml glucagon, 3.63 μg/ml hydrocortisone, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine. When the medium lacked trypsin inhibitor or atropine but contained the three hormones, the pigskin support began to be destroyed after 2 to 4 wk in culture. Thereafter, the cultured cells could not grow and survive on the digested pigskin. When 10⁻⁶ M atropine was added to the medium, amylase secretion from cultured cells and destruction of pigskin were inhibited completely but pancreas cells could not grow or survive. In contrast, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine permitted cell growth, permitted amylase secretion from the cultured acinar cells, and prevented the destruction of pigskin. Under these conditions pancreas cells migrated or grew or both from the organ bits onto the surface of the pigskin dermis and organoid aggregations formed. Hydrocortisone was needed to permit growth for more than 2 wk. Glucagon and insulin had additive effects. Light and electron microscopic observations indicated the culture of at least five kinds of cells, i.e., duct, acinar, centroacinar, endocrine, and mesenchymal. The majority of cultured cells were duct cells and acinar cells. There were few mesenchymal cells. Mouse pancreas cells were cultured for at least 12 wk by this method. This investigation was supported by PHS Grant CA 30220 awarded by the National Cancer Institute, DHHS, Grant 1203M awarded by the Council for Tobacco Research, Inc., and Grant RD-65 (for equipment) awarded by the American Cancer Society. Nude mice were provided by Dr. Wendall M. Farrow of Life Sciences, Inc., Resource Laboratory N01, CP6-1005 of the National Cancer Institute.     

Effects of androgen receptor and androgen on gene expression in prostate stromal fibroblasts and paracrine signaling to prostate cancer cells

The androgen receptor (AR) is expressed in a subset of prostate stromal cells and functional stromal cell AR is required for normal prostate developmental and influences the growth of prostate tumors. Although we are broadly aware of the specifics of the genomic actions of AR in prostate cancer cells, relatively little is known regarding the gene targets of functional AR in prostate stromal cells. Here, we describe a novel human prostate stromal cell model that enabled us to study the effects of AR on gene expression in these cells. The model involves a genetically manipulated variant of immortalized human WPMY-1 prostate stromal cells that overexpresses wildtype AR (WPMY-AR) at a level comparable to LNCaP cells and is responsive to dihydrotestosterone (DHT) stimulation. Use of WPMY-AR cells for gene expression profiling showed that the presence of AR, even in the absence of DHT, significantly altered the gene expression pattern of the cells compared to control (WPMY-Vec) cells. Treatment of WPMY-AR cells, but not WPMY-Vec control cells, with DHT resulted in further changes that affected the expression of 141 genes by 2-fold or greater compared to vehicle treated WPMY-AR cells. Remarkably, DHT significantly downregulated more genes than were upregulated but many of these changes reversed the initial effects of AR overexpression alone on individual genes. The genes most highly effected by DHT treatment were categorized based upon their role in cancer pathways or in cell signaling pathways (transforming growth factor-β, Wnt, Hedgehog and MAP Kinase) thought to be involved in stromal-epithelial crosstalk during prostate or prostate cancer development. DHT treatment of WPMY-AR cells was also sufficient to alter their paracrine potential for prostate cancer cells as conditioned medium from DHT-treated WPMY-AR significantly increased growth of LNCaP cells compared to DHT-treated WPMY-Vec cell conditioned medium.     

Primary culture of axolotl spinal cord ependymal cells

In order to examine the role of ependymal cells in the spinal cord regeneration of urodele amphibians, procedures were established to identify and culture these cells. Cell isolation and culture conditions were determined for ependymal cells from larval and adult axolotls (Ambystoma mexicanum). Dissociated cells prepared from intact spinal cords were cultured on fibronectin- or laminin-coated dishes. Dissociated cells attached more rapidly to fibronectin, but attached and spread on both fibronectin and laminin. Essentially pure populations of ependymal cells were obtained by removing 2 week old ependymal outgrowth from lesion sites of adult spinal cords. These ependymal outgrowths attached and grew only on fibronectin-coated dishes. Growth and trophic factors were tested to formulate a medium that would support ependymal cell proliferation. The necessary peptide hormones were PDGF, EGF, and insulin. TGF-beta(1) affected the organization of cell outgrowth. Initially, longterm culture required the presence of high levels of axolotl serum. Addition of purified bovine hemaglobin in the culture medium reduced the serum requirement. Outgrowth from expiants was subcultured by transferring groups of cells. Intrinsic markers were used to identify ependymal cells in culture. The ependymal cells have characteristic ring-shaped nucleoli in both intact axolotl spinal cords and in culture. Indirect immunofluorescence examination of intermediate filaments showed that ependymal cells were glial fibrillary acidic protein (GFAP) negative and vimentin positive in culture. Identification of dividing cells was made using (3)H-thymidine incorporation and autoradiography, and by the presence of mitotic figures in the cultured cells.     

Nullipotent Teratocarcinoma Cells Acquire the Pluripotency for Differentiation by Fusion with Somatic Cells

Abstract. By fusion of nullipotent embryonal carcinoma F9 cells with certain somatic cells freshly collected from differentiated tissues such as thymus and lens, pluripotent hybrid cell lines were obtained. They exhibited a wide spectrum of differentiation, including neural tubes, cartilages, skeletal muscles, ciliated epithelia and others, in solid tumors formed after injection into syngeneic mice. Cells from these tumors differentiated into several cell types when cultured in vitro. A possibility of the introduction of genes to code the factors for regulating differentiation into F9 cells by fusion is suggested.     

Haploid and diploid cell cultures from a haplo-diploid insect

Summary

This is the first report of haploid and diploid cell culture from the haplo-diploid parasitoid wasp, Mormoniella vitripennis. Cells were cultured from haploid and diploid wasps by collecting populations of eggs from virgin females (unfertilized, haploid, parthenogenetic eggs) and mated females (mostly fertilized, diploid eggs). Eggs were surface sterilized in 70% ethanol, followed by 50% Chlorox, and rinsed in phosphate buffered saline; larvae were allowed to hatch in culture. Larval cells were dissociated and cultured at 28°C in the presence of Grace's medium supplemented with fetal bovine serum. Most cells in the HMV (predominantly haploid) and DMV (predominantly diploid) cell cultures grew in suspension in the first week, formed monolayers of fibroblasts and epithelial cells by the second week in culture, and continued to grow in monolayers and vesicle-like structures for up to three months. Chromosome analysis of HMV. cells demonstrated over 70% haploid cells, with five chromosomes (N=5). The remainder were aneuploid. No diploid cells (2N= 10) were found in the HMV cell culture. Chromosome analysis of DMV cultures revealed 62% diploid, with ten chromosomes; 13% were haploid, with five chromosomes; the remainder were aneuploid. These data confirm that haploid and diploid cells can be cultured from a haplo-diploid insect species. The HMV cells which are predominantly haploid, and DMV cells which are predominantly diploid may be valuable models for the study of cellular and gene activity in haploid and diploid genetic milieux.     

Effects of dihydrotestosterone and hydroxyflutamide on androgen receptors in cultured human breast cancer cells (EVSA-T).

The purpose of our study was to evaluate the effects of 5 alpha-dihydrotestosterone (DHT) and hydroxyflutamide (HF), alone or in combination, on androgen receptor (AR) dynamics and on cellular growth in cultured breast cancer cells (EVSA-T). The incubation of cells with DHT increased the concentration of nuclear AR after 24 and 48 h. HF was also able to promote the nuclear accumulation of AR after 24 and 48 h of treatment. When HF-treated cells are incubated with DHT, the nuclear AR concentration is lower than that found in cells treated with DHT alone. We conclude that HF acts by increasing nuclear accumulation of receptor-antiandrogen complexes. Moreover, DHT stimulates cell growth while HF has an inhibitory effect. Thymidine incorporation in cells also increased after DHT treatment and decreased after HF incubation. The HF-induced inhibition of cell growth persisted both after renewal of the medium and after the addition of DHT to cultures. It may be hypothesized that either DHT is converted to inactive metabolites or that HF exerts a persistent inhibitory effect. In the latter case, the antiandrogen action of HF could be exerted by retention of high levels of antiandrogen in cells or by such a depressed protein synthesis that the renewal of growth is slower than the 48 h period studied.     

Multiple binding components for methyltrienolone in canine prostatic epithelial cells

Using a whole cell assay system, the androgen binding capacity of canine prostatic epithelial cells was evaluated in relation to their function. Radiolabeled Methyltrienolone (R1881) was used as the ligand in the presence of an excess of Triamcinolone acetonide and the amount of [3H]R1881 bound to the cells at equilibrium was determined by either displacement or saturation studies. With immature cells in culture (3 days of attachment), displacement analysis revealed the presence of high affinity binding sites which were also present in cells cultured for 10 days. With freshly dispersed prostatic cells (mostly secretory epithelial cells) as well as with older cells in culture (17 and 24 days), only less specific binding sites were observed with both unlabeled R1881 and/or dihydrotesterone (DHT). In contrast, only the high affinity androgen receptor (AR) was present in cytosolic extracts prepared from normal glands. Displacement studies performed with cultured cells at different stages of growth also showed that the basal level as well as the degree of low affinity binding increased during the maturation of non-proliferating cells. The presence of multiple binding components was demonstrated by saturation studies performed with either cultured or freshly dispersed cells. The first component, that was saturated at 5 nM of [3H]R1881, was due to AR while the other two binding components, showing positive-cooperativity (Hill coefficients of 1.90 and 5.07, respectively), were saturated at concentrations of 15 and 30 nM of [3H]R1881. In contrast, the Hill coefficient for the AR was 0.88 indicating the presence of an independent component. It was calculated that only 11.4% of the total uptake of R1881 was attributed to AR binding, suggesting that the remainder may represent an intracellular pool of androgens. Thus, a whole cell binding assay represents a dynamic system for the detection, by saturation studies, of binding components that are not revealed using the conventional displacement studies or cell-free systems. It is proposed that these acceptor sites may play a role in differentiated prostatic function rather than in cell proliferation.     

Distribution of concanavalin A binding sites on the surface of dissociated rat submandibular gland acinar cells.

The submandibular glands of 4-week-old rats were dissociated by a procedure involving digestions with collagenase and hyaluronidase, chelation of divalent cations and mechanical force. A suspension of single cells was obtained in low yield by centrifugation in a Ficoll-containing medium. Immediately after dissociation and after a culture period of 16-18 hr the dissociated cells were tested for agglutinability by concanavalin A (Con A). Using ferritin (tfer)-conjugated Con A the lectin binding by the isolated acinar cells was also studied. The dissociated cells were agglutinated by low concentrations of Con A and bound Fer-Con A molecules on their entire surface without any indication of polarization of the cell membrane. There was a considerable cell to cell variation in the amount of Fer-Con A binding which was, in general, sparse and patchy. The contact surfaces between agglutinated cells revealed a dense binding of Fer-Con A molecules irrespective of the types of cells participating in the agglutination reaction. Cells cultured for 16-18 hr were no longer agglutinated by Con A. As compared to the freshly dissociated cells the cultured acinar cells revealed a more uniform and denser binding of Fer-Con A molecules. Furthermore, there were more lectin molecules bound to the cell surface corresponding to the basal part of the cell, where the nucleus and most of the rough surface endoplasmic reticulum were located, than to the apical cell surface. It is suggested that the higher density of lectin-binding sites on the cell surface in the vicinity of the cisternae of the rough endoplasmic reticulum indicates insertion sites of newly synthesized membrane glycoproteins.