Propagation of mouse mammary tumor cell lines and production of mouse mammary tumor virus in a serum-free medium.

Five different mouse mammary tumor cell lines were propagated in a serum free medium. Evaluation of growth characteristics, including logarithmic growth, cell population increase, protein production and days to confluency, showed serum-free medium comparable to serum-containing medium. Mouse mammary tumor virus expression and production, in C3H and GR tumor cell lines, as determined by virus particle counting and RNA dependent DNA polymerase assays, subsequent to dexamethasone stimulation revealed equivalent to higher levels of virus in serum-free medium as compared to serum-containing medium.     

Demonstration of components of serum-free culture medium effecting maximum in vitro expression of mouse mammary tumor virus

Summary By using a chemically defined serum-free (SF) medium for propagation of Mm5mtc₁ mouse adenocarcinoma cell cultures and clonal derivatives, medium components including hormones, glucose and individual amino acids were evaluated as to modulation of mouse mammary tumor virus (MMTV) production. Insulin, hydrocortisone and dexamethasen each increased MMTV production on a per cell basis over constitutive expression that ocures in SF medium devoid of hormones. Maximum production occurred when all three hormones were present. Hormone-stimulated virus expression also was influenced by glucose concentration. Cell growth and maximum MMTV expression increased when thyroxine, asparagine, proline and serine were omitted from the medium formulation. The resulting modified SF medium provides and ideal system for the propagation of high MMTV-producer clones and for the study of the biochemical regulation of MMTV expression. This work was supported in part by the Virus Cancer Program, Contract No. N01-CO-25423, National Cancer Institute, NIH, Bethesda, Maryland.     

Cell culture factors influencing in vitro expression of mouse mammary tumor virus

Summary Several cell culture factors were found to influence in vitro expression of mouse mammary tumor virus (MMTV) in the mouse adenocarcinoma cell line Mm5mt/c₁. Cells were propagated in a variety of commercially available cell culture media to which dexamethasone (DXM) was added as a stimulator of MMTV production. Culture seeding density, culture medium type, and glucose concentration each influenced MMTV production when expressed on a per cell basis. Maximum cell growth occurred in cultures grown in RPMI-1640 medium containing insulin. Those media which provided good cell growth were not necessarily optimal for virus expression. Addition of insulin did not stimulate MMTV synthesis although dexamethasone alone was stimulatory in all media used; however, maximum MMTV expression was obtained when dexamethasone and insulin were used in concert. Equivalent levels of MMTV-specific cell membrane antigen, MMTV-specific protein, and virus particles were produced at incubation temperatures of 32°, 34° or 37° C; however, higher levels of virus-related RNA-dependent DNA polymerase (RDDP) activity were recovered from cultures incubated at 32° and 34° C than at 37° C. Decreased levels of RDDP were attributed to enzyme thermolability at 37° C incubation. Research sponsored by the National Cancer Institute under Contract No. N01-CO-25423 with Litton Bionetics, Inc., and Contract No. N01-CP-33253 with the University of California.     

Increased yield of mouse mammary tumor virus (MMTV) by cultivation of monolayer-derived mammary tumor cells in suspension

Summary The MJY-alpha epithelial-like mammary tumor cell line was adapted for cultivation in suspension using a shaker culture technique. Replication of suspension (MJY-beta) cells was more sensitive than monolayer cells to decreases in the concentration of serum in the medium. Comparison of amino acid incoerporation and lactate production rates revealed additional differences between monolayer and suspension cultures. In addition, growth in susfpension resulted in 10- to 400-fold increases in mouse mammary tumor virus (MMTV) production by the mammary tumor cells. Incrases in MMTV yield were detected within 48 h of culture initiation and MMTV production remained elevated throughout 20 cell passages in suspension. Exposure of MJY-beta cells to 14 μM hydrocorticone further increased MMTV yield two-to five-fold. The MJY-beta suspension cultures demonstrated that these epithelial-like cells do not require attachment to a solid substrate for replication or for MMTV production. Loss of structural polarization associated with growth as a monolayer resulted in stimulation of MMTV production greater than and independent of steroid exposure. This work was supported by the T. J. Martell Foundation for Cancer and Leukemia Research and by USPHS grant 5P-30CA23102. F. M. is a trainee on MSTP grant GM07280 from the National Institute of Health. This work was submitted in partial fullfillment of the requirements for the Ph. D. degree (F. M.).     

Serum-Free Suspension Culture of MDCK Cells for Production of Influenza H1N1 Vaccines

Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID₅₀) titers of the two cell lines reached 4.51×10⁶ cells/mL, 2.94Log₁₀(HAU/50 μL) and 8.49Log₁₀(virions/mL), and 5.97×10⁶ cells/mL, 3.88Log₁₀(HAU/50 μL), and 10.34Log₁₀(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted.     

Expression of the androgen-dependent MMTV-specific orf gene in Shionogi 115 mouse mammary tumor cells

The Shionogi 115 (S115) mouse mammary tumor cells express the MMTV-specific 1.7 kb mRNA (orf) at a high level in the presence of androgens. In lymphoid cells the orf-gene encodes a superantigen which has an important role in establishing self-tolerance but in mammary and breast cancer cells the function of the orf gene is unclear. In the present work we studied the expression of the S115 mammary tumor cell orf sequence and its role in the androgen regulated growth of S115 cells. The cloning and sequencing of the cDNA specific for the 1.7 kb mRNA from the S115 mouse mammary tumor cells revealed a 990 bp DNA sequence with a 99.8% homology to the Mtv-17 proviral strain. There was a difference of only one amino acid (isoleu-tyr) in the coding region. A peptide was synthesized according to the hypervariable C-terminal part of the predicted protein and used to raise a rabbit antiserum. The anti-S115-orf antiserum immunoprecipitated an approximately 45 kDa protein from the metabolically labeled S115 cell lysates. In order to analyze the putative functions of the protein, the orf-sequence was linked to MoMLV-LTR and to the human ß-actin promoter in the mammalian expression vectors pLTRpoly and pHßAPr-1-neo, respectively, and transfected into NIH3T3 and S115 cells. NIH3T3 transfectants expressing orf mRNA did not show a transformed phenotype in vitro. The S115 orf transfectants proliferated somewhat more slowly than the vector transfected control cells in cell culture, both in the presence or absence of androgen, but there was no obvious change in the phenotype of S115 cells or in expression of the fibroblast growth factor 8 (FGF-8). This factor is activated by Mtv-6 integration and mediates androgen effects in these cells. Unexpectedly, however, the formation of tumors by S115 orf cells in nude mice was considerably prolonged and tumor growth retarded when compared with vector transfected control or parent S115 cells. The results suggest that MMTV-orf can be functional in breast cancer cells but the mechanism of the growth repressive effect in mammary tumor remains to be analyzed.     

Endogenous prostaglandin production by established cultures of neoplastic rat mammary epithelial cells

Summary The production and release of prostaglandins (PGs) into the growth medium by established cultures of neoplastic, mammary epithelial cells derived from (a)N-nitrosomethyl-urea (NMU)-induced and (b) 7,12-dimethylbenz(a) anthracene (DMBA)-induced mammary tumors, was assessed using radioimmunoassay techniques. Prostaglandin production was determined, to a considerable extent, by in vitro conditions and the tumor line analyzed. In medium supplemented with bovine calf serum (10%), NMU cells synthesized and released nanogram quantities of PGF₂, PGE₁, and PGF_2α (6.7, 4.7, and 1.7 ng/10⁶ cells per 48 h, respectively). Concentrations of the two stable protanoid metabolites, 6-keto-PGF_1α and TXB₂, were indistinguishable from controls. In cells derived from the DMBA-induced tumor (RBA cells), no net production of immunoreactive PGs was detected. In contrast, in media supplemented with fetal bovine serum (10%), both RBA and NMU cells synthesized and released nanogram quantities of PGE₂ (1 and 4 ng/10⁶ cells per 48 h, respectively). PGE₂ production by both NMU and RBA cells was inhibited by ibuprofen, an inhibitor of cyclooxygenase (EC 1. 14. 99.1). The pattern of PG inhibition by ibuprofen differed in the two cell lines. In NMU cells, a linear dose-response inhibitory pattern was discernable, whereas in RBA cells a biphasic pattern was observed; PGE₂ levels incresed at low concentration of ibuprofen and then decreased at higher concentration. At 100 μg/ml ibuprofen, PG synthesis and release was inhibited by 90 and 100% and cell growth by 64 and 66% in NMU and RBA cells, respecively. There was no obvinous dse-response relationship between ibuprofen concentration and cell growth inhibition in either cell line. These results underline the importance of the serum component of growth medium when analyzing PG production in vitro and suggest that the epithelial cell components of experimental mammary tumors are capable of producing physiogically relevant amounts of PGs. This work was supported by Grant CA 29602 from the National Cancer Institute, Bethesda, MD, and Grant PDT-208 from the American Cancer Society.     

A human breast tumor cell line (BT-474) that supports mouse mammary tumor virus replication

Summary A human breast tumor cell line BT-474 derived from an invasive ductal carcinoma was experimentally infected in vitro with a mouse mammary tumor virus from the RIII strain (RIII-MuMTV). The virus that replicated in the human cells was characterized as a mouse virus by immunofluorescence, electron microscopy and the presence of a specific RNA-directed DNA polymerase. The cells themselves were human as per the karyotype and isoenzyme migration patterns. It is concluded that human cells are susceptible to the mouse mammary tumor virus and can, eventually, support its replication. This work was supported by USPHS Grant CA-08515 from the National Cancer Institute and by NIH Contract N01-CP-81003.     

Demonstration of components of serum-free culture medium effecting maximum in vitro expression of mouse mammary tumor virus.

By using a chemically defined serum-free (SF) medium for propagation of Mm5mtc1 mouse adenocarcinoma cell cultures and clonal derivatives, medium components including hormones, glucose and individual amino acids were evaluated as to modulation of mouse mammary tumor virus (MMTV) porduction. Insulin, hydrocortisone and dexamethasone each increased MMTV production on a per cell basis over constitutive expression that occurs in SF medium devoid of hormones. Maximum production occurred when all three hormones were present. Hormone-stimulated virus expression also was influenced by glucose concentration. Cell growth and maximum MMTV expression increased when thyroxine, asparagine, proline and serine were omitted from the medium formulation. The resulting modified SF medium provides and ideal system for the propagation of high MMTV-producer clones and for the study of the biochemical regulation of MMTV expression.     

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.     

Growth of MTW9/PL2 estrogen-responsive rat mammary tumor cells in hormonally defined serum-free media

Summary Growth of the estrogen-responsive MTW9/PL2 rat mammary tumor cells was demonstrated in serum-free defined medium (designated DDM-1) formulated with F12-DME (1:1 vol/vol) supplemented with 15 mM HEPES pH 7.4 insulin 10 μg/ml, transferrin 10 μg/ml, sodium selenite 10 ng/ml, triiodo-l-thyronine 0.3 nM, phosphoethanolamine 5 μM, epidermal growth factor (20 ng/ml), 17 β-estradiol 2 nM, and bovine serum albumin 20 μg/ml. In DDM-1, the growth rate was about one-half that seen in serum-containing medium. When ethanolamine (50 μM), glutathione (20 μg/ml), and linoleic acid/bovine serum albumin (150 μg/ml) were added (formulation DDM-2), the growth rate was 80% of serum-containing medium and not seed-density dependent. Deletion of estradiol from DDM-1 or DDM-2 had no effect on growth rate. Also, cells grown in steroid hormone deficient medium for 4 mo. continued to form estrogen-responsive tumors in rats as did cells cultured for the same period in 2 nM estradiol. To investigate autocrine growth factor secretion, a third medium (DDM-3) was prepared by deleting insulin, epidermal growth factor, phosphoethanolamine, estradiol, and both forms of bovine serum albumin from DDM-2. Growth in mitogen-free medium equaled 86% of the serum-stimulated rate and was seed-density dependent; phenol red deletion from DDM-3 had no effect on growth rate. Evidence presented suggests that autocrine factors stimulate growth of the MTW9/PL2 cells in DDM-3, and that this secretion may support the growth of estrogen-responsive cells in culture in the absence of steroid hormone. This work was supported by National Cancer Institute grants CA-38024 and CA-26617 and American Cancer Society grant BC255.     

Evaluation of the new serum-free medium (MDSS2) for the production of different biologicals: Use of various cell lines

The presence of serum in cell culture raises safety problems for the production of biologicals, thus a new serum-free medium (MDSS2) was developed. The evaluation of this medium for the growth of different cell lines (BHK-21 C13, BSR and Vero) has shown that cells grew in this medium similarly to standard serum-containing medium, independently of the culture system used: in static (as monolayer) as well as in agitated systems (in suspension in spinner and perfusion reactors). BHK-21 and BSR cells grew as aggregate cultures and could proliferate in both static and agitated culture systems. Vero cells stayed attached to a substrate and proliferated equally in static and in agitated microcarrier-culture systems. The cell densities obtained with BHK-21 cells depended only on the culture system used. They ranged from 2–3×10⁶ to 6–12×10⁶ cells per ml for static batch and perfusion reactor cultures respectively. The cell concentration was 3 to 6 times higher than in classical cultures performed in serum-containing medium. The cell densities obtained with Vero cells were indistinguishable from those obtained in serum-containing medium, whatever the cell culture system used. These cell lines have been used for the production of rabies virus. With respect to BHK-21 and BSR, similar production rates of rabies glycoprotein have been found as in the standard roller bottle process. The production of rabies virus and of viral glycoprotein by Vero cells cultivated in serum-free medium was augmented 1.5-fold and 2.5-fold, respectively, when compared to serum-containing medium.A recombinant BHK-21 cell line, producing human IL-2, can also proliferate in MDSS2, after addition of insulin. The specific IL-2 production rate was augmented 3–4 fold in comparison to serum-containing medium.For the cells tested, the MDSS2 serum-free medium is a good growth and production medium. Its use for cultivating other cell lines and/or for the production of other biologicals is discussed.     

High prevalence of mouse mammary tumor virus-like gene sequences in breast cancer samples of Iranian women

Mouse mammary tumor virus (MMTV) is considered to be responsible for breast tumor development in mice. In several instances sequences homologous to the genome of this virus have been reported in human breast cancer samples.

Here we aimed to evaluate MMTV involvement in human breast cancer development. DNA was extracted from 118 formalin fixed and paraffin embedded malignant (n = 59) and benign (n = 59) breast lesions. Using two sets of outer and inner MMTV-like envelope specific primers, in a nested PCR setup, MMTV genome was detected in 19 samples out of 59 (%32.2) cases of breast carcinomas, while in non-malignant breast tissue samples, only 3 samples out of 59 (%5) were positive. The difference was statistically highly significant (p < 0.001). Alignment of PCR amplified sequences with BR6, GR and C3H mouse mammary tumor virus strains emerged to be around 99% identical which is indicative of tumor tissue infection by an exogenous mouse MMTV genome.     

Growth factor interactions between mouse mammary cell lines cocultured in collagen gels

Summary Three related mouse mammary cell lines were cultured in collagen gels and assayed for growth factor responsiveness and interaction via soluble factors. The CL-S1 cell line is nontumorigenic and grows poorly in collagen gel culture. The +SA and −SA cell lines exhibit different degrees of malignant behavior in vivo and have different growth properties in vitro. In collagen gel culture, +SA growth was stimulated by serum but not by epidermal growth factor (EGF), whereas both serum and EGF were required for optimal growth of −SA cells of early passage number as well as CL-S1 cells. −SA cells of later passage repeatedly exhibited a change so as to no longer require serum while retaining EGF responsiveness. [¹²⁵I]EGF binding analyses indicated that CL-S1 cells bound EGF with less affinity than did −SA cells whereas +SA cells bound almost to ligand. When cell lines were maintained in separate collagen gels but shared the same culture medium, growth of +SA or −SA cells was slightly enhanced in the presence of CL-S1 cells and −SA cell growth was enhanced by the presence of +SA cells. Using the normal rat kidney fibroblast line NRK (clone 49F) as an indicator, serum-containing conditioned media from each cell line and from each pair of cell lines cultured in collagen gels were tested for transforming growth factor (TGF) activity. Both the −SA and CL-S1 lines tested positive for TGF-α production and possibly released a TGF-β activity. These results suggest mechanisms by which cell populations in and around tumors can modify one another’s growth characteristics. The work was supported by a grant from the American Institute for Cancer Research, by American Cancer Society Institutional grant IN-119, by funds from the Poncin Trust (Seattle-First National Bank), and by grants CA-39611 and CA46885 from the National Institutes of Health, Bethesda, MD.     

Inappropriate P-cadherin expression in the mouse mammary epithelium is compatible with normal mammary gland function

Cadherins comprise a family of cell-cell adhesion proteins critical to the architecture and function of tissues. Expression of family members E-, N-, and P-cadherin is regulated in a spatial and temporal fashion in the developing and adult organism. Using in vivo and in vitro experimental systems, perturbation of cadherin expression by genetic deletion, overexpression, mutant dominant-negative constructs, and, to a lesser degree, expression of an inappropriate cadherin have all been shown to alter embryogenesis, tissue architecture, and cell behavior. Here we studied how expression of an inappropriate cadherin affects the adult mouse mammary gland. Human P-cadherin was expressed in mammary epithelial cells under control of the mouse mammary tumor virus (MMTV) promoter, and the effect on mammary gland behavior was studied. Typically, E-cadherin is expressed by mammary epithelial cells, whereas P-cadherin is found in myoepithelial cells and cap cells of the ductal terminal end bud. However, breast cancers frequently express P-cadherin, even though they are thought to arise from epithelial cells, and it is a marker of poor prognosis. We developed two independent transgenic mouse lines that exhibited high levels of P-cadherin protein expression in the mammary epithelium. P-cadherin was detected in most, but not all, luminal epithelial cells, and was appropriately localized to cell-cell borders. It was detected in the mammary glands of virgin, pregnant, lactating, post-lactation, and aged parous female mice. Despite the robust and widespread expression of an inappropriate cadherin, no effect was observed on mammary gland morphogenesis, architecture, lactation, or involution in transgenic mice compared to wild-type mice. No mammary tumors formed spontaneously in either wild-type or transgenic mice. Moreover, mammary tumors induced by the neu oncogene, which was introduced by a breeding strategy, showed no differences between mice with or without hP-cadherin. Surprisingly, however, none of the tumors expressed hP-cadherin protein. Together, our studies show no apparent effect on adult mammary gland or tumor behavior by inappropriate expression of P-cadherin in normal mammary epithelial cells.     

Establishment and characterization of a new murine mammary tumor cell line,balb/c-MC

Summary A new murine mammary tumor cell line (BALB/c-MC) was established from a spontaneous mammary tumor in a 17-mo.-old female mouse of the low mammary cancer strain BALB/cHe. The cell line was derived from a papillary adenocarcinoma. In monolayer culture the line exhibits a pavementlike arrangement of cells and forms “domes” or “hemicysts” as the cells become confluent. The cell line rapidly forms tumors when transplanted into young syngeneic BALB/cHe mice. The subcutaneous injection of 10⁶ cells resulted in the development of mammary tumors (typical papillary adenocarcinomas) in 33 of 37 (87% recipients within 2 to 3 mo. after injection. These mammary tumors also metastasize to lung [14 of 33 (42%) of recipients] during this time. The number of chromosomes in this cell line is hyperdiploid (average of 43, range 39 to 44).     

Primary culture of mouse mammary tumor epithelial cells embedded in collagen gels

Summary Mammary tumor epithelial cells from BALB/cfC3H mice were dispersely embedded inside the collagen gels in Ham's F-12 medium containing horse serum. A sustained cell growth leading to a 5- to 10-fold increase in cell number over initial level was observed in less than 2 weeks. The extent of this growth was found to be dependent on serum concentration. However, addition of various protein and steroid hormones, both singly and in combination, to low-serum-containing medium failed to achieve a comparable level of growth to that promoted by higher serum concentration. Mammary tumor cells can now be consistently propagated in primary culture. This investigation was supported by Grants CA05388 and CA09041 awarded by the National Cancer Institute, Department of Health, Education and Welfare, and by cancer research funds of the University of California.     

Characterization of a new spontaneously developed murine mammary adenocarcinoma in syngeneic BALB/c hosts

Summary A mouse mammary tumor cell line, desingated JC, has been established from a spontaneously developed primary adenocarcinoma of an aged virgin female BALB/c mouse. Isoenzyme analyses including glucose-6-phosphate dehydrogenase, lactate dehydrogenase, and peptidase proved that this cell line is of murine origin and devoid of contamination from other species. Karyotyping revealed that the number of chromosome ranged from 26 to 100, with a modal number of 40. Electron microscopic examination detected the presence of tonofilament and desmosomes confirming its epithelial nature. In addition, no type B or C virus particle was detected, although intracysternal A particle was observed occasionally. Tumorigenicity in immunocompetent syngeneic hosts was easily established by s.c., i.p., and i.v. injection of viable JC tumor cells. A very weak immunogenicity of the JC tumor was demonstrated through its immunization-challenging on syngeneic immunocompetent hosts. Although no rejection of JC tumor was noted, a significant prolongation for the incubation period before an obvious and palpable tumor growth was detected between the experimental and the control animals. Development of a concomitant immunity was also detected. The JC tumor represents a valuable murine mammary tumor model which is different from other available models because of its unique origin, absence of virus particles, very weak immunogenicity, and high tumorigenicity in syngeneic hosts. The cell line has been maintained for more than 5 yr and has been used for experimental immunotherapy in our laboratory. This work was supported by a research grant IM-416, awarded by the American Cancer Society, Atlanta, Georgia.     

Cell culture factors influencing in vitro expression of mouse mammary tumor virus.

Several cell culture factors were found to influence in vitro expression of mouse mammary tumor virus (MMTV) in the mouse adenocarcinoma cell line Mm5mt/c1. Cells were propagated in a variety of commercially available cell culture media to which dexamethasone (DXM) was added as a stimulator of MMTV production. Culture seeding density, culture medium type, and glucose concentration each influenced MMTV production when expressed on a per cell basis. Maximum cell growth occurred in cultures grown in RPMI-1640 medium containing insulin. Those media which provided good cell growth were not necessarily optimal for virus expression. Addition of insulin did not stimulate MMTV synthesis although dexamethasone alone was stimulatory in all media used; however, maximum MMTV expression was obtained when dexamethasone and insulin were used in concert. Equivalent levels of MMTV-specific cell membrane antigen, MMTV-specific protein, and virus particles were produced at incubation temperatures of 32 degrees, 34 degrees or 37 degrees C; however, higher levels of virus-related RNA-dependent DNA polymerase (RDDP) activity were recovered from cultures incubated at 32 degrees and 34 degrees C than at 37 degrees C. Decreased levels of RDDP were attributed to enzyme thermolability at 37 degrees C incubation.