Different hormonal requirements for androgen-independent growth of normal and tumor epithelial cells from rat prostate

Summary The proliferation of isolated normal prostate epithelial cells from rat and man is androgen-independent and requires cholera toxin, insulin, dexamethasone, epidermal growth factor (EGF) and one or more polypeptide factors that are concentrated in bovine neural tissue. The active agents in the neural tissue extract are heparin-binding polypeptides (prostatropins), the predominant form of which has a molecular weight of 17400 and an acetylalanine at the aminoterminus. Prostatropins supported a half-maximal increase in normal prostate epithelial cell number at 50 picomolar. The proliferation of primary and serially-cultured epithelial cells from androgen-responsive Dunning R3327 rat prostate tumors was also androgen-independent, but exhibited dramatic alterations in response to hormones that stimulated normal cell proliferation. At low cell density, androgen-independent growth of isolated tumor-derived epithelial cells was independent on cholera toxin, was stimulated by dexamethasone, required insulin andeither EGFor prostatropin. The presence of either EGF or prostatropin masked the response to the other factor. In the absence of EGF, purified prostatropins supported a half-maximal increase in tumor cell number at 7 picomolar. Endogenous production of EGF-like and prostatropin-like factors or both was suggested by the reduced requirement for EGF and prostatropin at high prostate tumor cell density. These results suggest that anti-hormonal therapies against prostate tumor growth should be based on intervention with the activity of insulin (or insulin-like factors) or simultaneous intervention with both EGF and prostatropin (or their homologues). This work was supported by NIH grants CA 37589 and HL 33847, and grant 1718 from the Council for Tobacco Research. Editor’s Statement This paper is the first report of the comparison of the hormone requirements of primary cultures of normal and tumor prostate epithelial cells from the same system.     

Ultraviolet radiation-induced neoplastic transformation of normal human cells,in vitro

Human foreskin cell cultures in scheduled DNA synthesis (S phase) of the cell cycle were exposed to UV irradiation at a dose of 10 J · m^?2 in the presence of insulin. These treated cell populations, when selectively passaged in a high amino acid supplemented complete growth medium (CM) after 20 Dulbecco's phosphate buffered saline (pH 6.8) (PDL), were able to be grown in soft agar. These treated cell populations were also grown in 1% serum supplemented growth medium and at 41°C in 10% serum supplemented growth medium. Cell populations 4–5 PDL after treatment exhibited altered colony morphology and altered lectin agglutination profiles but would not grow in soft agar. These events appeared to be associated with the early stages in the expression phase of the transformed phenotype. After 20 PDL, we observed that these cells would grow in soft agar at a frequency of 20 colonies/10⁵ cells seeded in soft agar. The cell populations derived from these colonies, when propagated and injected into the nude mice, formed myxofibromas at the injection sites rather than the type of tumor (fibrosarcoma) previously described for chemical carcinogen-induced neoplasms.     

Growth regulation of serum-free cultures of epithelial cells from normal human buccal mucosa

Summary Human buccal epithelial cells have been reared from explants maintained in supplemented MCDB 153 medium. Primary epithelial outgrowths show typical structural features and uniformly express keratins; subunit analyses demonstrate expression of keratins 5, 6, 14, 16/17, and 19. The cells exhibit up to 6% colony forming efficiency and divide at about 0.8 population doublings per day on fibronectin/collagen-coated dishes at clonal density. Studies of markers of proliferation and differentiation in buccal epithelial cells indicate that epidermal growth factor, cholera toxin, retinoic acid, and pituitary extract each exhibit a distinctive ability to enhance growth and variably affect cell migration and cell surface area. Transforming growth factorβ-1 inhibits growth and increases surface area without affecting migration, involucrin expression, and cross-linked envelope formation. Moreover, exposure of cells to fetal bovine serum, the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate or an elevated Ca²⁺ concentration (from 0.1 to 1 mM) inhibits growth and induces squamous differentiation as indicated by inhibition of migration, increases in surface area, involucrin expression, or formation of cross-linked envelopes. The results show that epithelial cells can be reproducibly derived from explant cultures of human buccal mucosa specimens and the cells transferred under serum-free conditions. Buccal epithelial cells in culture undergo a pattern of growth and differentiation that mimics parakeratinization in vivo and variably respond to several agents shown to modulate growth of cells that originate from other types of epithelia. This work was supported in part by grants from the Swedish National Board of Laboratory Animals, the Swedish Medical Research Council, the Swedish Natural Science Research Council, the Swedish Fund for Scientific Research Without Animal Experiments, the Swedish Cancer Society, the Swedish Tobacco Company, and Lions Club International, Djurg?rden, Stockholm, Sweden.     

Growth of normal and neoplastic rat pleural mesothelial cells in the presence of conditioned medium from neoplastic mesothelial cells

Several transformed cells have been demonstrated to secrete growth factors. We studied the effect of conditioned medium from neoplastic rat pleural mesothelial cells on normal and neoplastic mesothelial cell growth. The results showed that the concentrated conditioned medium stimulated neoplastic mesothelial cell growth but inhibited reversibly normal mesothelial cell growth.     

Proteoglycan synthesis by normal and neoplastic human transitional epithelial cells

Metabolically 35S-labeled proteoglycans were isolated from cell-associated matrices and media of confluent cultures of human normal transitional epithelial cells and HCV-29T transitional carcinoma cells. On Sepharose CL-4B columns, the cell-associated proteoglycans synthesized from both cell types separated into three identical size classes, termed CI, CII, and CIII. Normal epithelial cell C-fractions eluted in a 22:34:45 proportion and contained 64%, 64%, and 72% heparan sulfate, whereas corresponding HCV-29T fractions eluted in a 29:11:60 proportion, and contained 91%, 77%, and 70% heparan sulfate, respectively. Medium proteoglycans from normal cells separated into two size classes in a proportion of 6:94 and were composed of 35% and 50% heparan sulfate. HCV-29T medium contained only one size class of proteoglycans consisting of 23% heparan sulfate. The remaining percentages were accounted for by chondroitin/dermatan sulfate. On isopycnic CsCl gradients, proteoglycan fractions from normal cells had buoyant densities that were higher than the corresponding fractions from HCV-29T cells. DEAE-Sephacel chromatography showed that cell and medium associated heparan sulfate from HCV-29T cells was consistently of lower charge density (undersulfated) than that from normal epithelial cells. In contrast, the chondroitin/dermatan sulfate of HCV-29T was of a charge density similar to that of normal cells. These as well as other structural and compositional differences in the proteoglycan may account, at least in part, for the altered behavioral traits of highly invasive carcinoma cells.     

Growth of normal human mammary cells in culture

Summary Reduction mammoplasty tissue was used to obtain short-term cultures of human epithelial cell populations. Digestion of tissue with collagenase and hyaluronidase resulted in cell clusters (organoids) resembling ductal and alveolar structures; these could be separated by filtration from the stromal components. Epithelial outgrowth from these organoids was greatly enhanced by the addition of conditioned medium from other human epithelial and myoepithelial cell lines. Additionally, the mammary epithelial growth was stimulated by insulin, hydrocortisone, epidermal growth factor, and steroid hormones. With this enriched nutritional environment, active cell division could be maintained for 1 to 3 months and cells could be serially subcultured 1 to 4 times. This research was supported by Grant PDT-72 from the American Cancer Society and Grant CP-70510 from the National Institutes of Health.     

Plasticity of epithelial cells derived from human normal and ADPKD kidneys in primary cultures

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation initiated by dedifferentiation and proliferation of renal tubular epithelial cells. Renal tubular epithelial cells (RTC, derived from normal kidney tissue) in primary cultures exhibit both homogeneous expression of γ-glutamyl transferase and low molecular weight cytokeratin, two different markers for proximal and distal renal epithelial cells, respectively. RTC in cultures also abnormally express the dedifferentiation markers vimentin and PAX-2, which are proteins normally expressed in epithelial cells lining cysts in ADPKD kidneys but not tubular cells in normal kidneys. In contrast, different cultures of cystic epithelial cells (CEC, derived from the cysts walls of polycystic kidneys) display variable expression of cytokeratin, γ-glutamyl transferase, and PAX-2, but a constant level of vimentin. Importantly, RTC and CEC exhibit the capacity to convert to their respective original structures by forming tubules and cysts, respectively, when cultured in a three-dimensional gel matrix, whereas HK-2, LLC-PK1, and MDCK renal epithelial cell lines form cell aggregates or cysts. Our study demonstrates that the marker expression of the various epithelial cell types is not highly stable in primary cultures. Their modulation is different in cells originating from normal and ADPKD kidneys and in cells cultured in monolayer and three-dimensions. These results indicate the plasticity of epithelial cells that display a mixed epithelial/dedifferentiated/mesenchymal phenotype during their expansion in culture. However, RTC and CEC morphogenic epithelial properties in three-dimensional cultures are similar to those in vivo. Thus, this model is useful for studying the mechanisms leading to tubulogenesis and cystogenesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by a grant from The Polycystic Kidney Foundation. We gratefully acknowledge the support of the Children’s Medical Research Institute and Children’s Miracle Network Foundation.     

Growth and characterization of epithelial cells from normal human uterine ectocervix and endocervix

Summary The human uterine cervix consists of an endocervical canal lined with a single layer of columnar mucus-secreting cells and an outer ectocervix covered by a stratified squamous epithelium. We report here the culture of human endocervical epithelial cells (HEnE) and human ectocervical epithelial cells (HEcE) in serum-free medium (KGM). Both HEnE and HEcE cultures were composed of keratinocytelike cells which formed desmosomal contacts and stratified in the presence of high concentrations of calcium ions. Cells with a pleomorphic epithelial morphology were observed in HEnE cultures, but not in HEcE cultures. Keratin 18, which is characteristic of endocervix in vivo, was detected by indirect immunofluorescent staining in all HEnE cells but was never detected in cultured HEcE. HEcE expressed keratin 13 which is characteristic of ectocervix in vivo. Although keratin 13 was never detected in primary HEnE cultures, it was expressed in passaged HEnE cultures grown in medium with high concentrations of calcium and in late passage HEnE cultures. HEnE underwent an average of 15.1 population doublings during serial culture. Mean colony-forming efficiency during Passages 2 to 3 was 14.7% and mean population doubling time was 17.8 h. HEcE cultures underwent significantly more population doublings (29.0) than HEnE cultures, whereas colony-forming efficiencies and doubling times were similar to those determined for HEnE. HEnE and HEcE cells may be useful in developing in vitro models of cervical squamous metaplasia and for exploring the interactions between target cell differentiation, carcinogens, and papillomaviruses in the development of cervical neoplasia. This study was supported in part by the Rush University Committee on Research and by the Lester B. and Francis Knight and the S. Charles and Marsha Papageorge research funds.     

Growth factor binding and bioactivity in human kidney epithelial cell cultures

Summary Insulinlike growth factors (IGF) and epidermal growth factor (EGF) are produced in renal tissue, as are specific receptors for these hormones. To evaluate the significance of these observations to regulation of renal tubular cell proliferation, we have examined the interaction of IGF and EGF with cultured human proximal tubular epithelial cells (HPT). HPT cells showed specific binding of IGF-1, insulin, and EGF. IGF-1 binding was inhibited by antibody to the type 1 IGF receptor (α-IR3). Insulin receptors and type 1 IGF receptors were identified by bifunctional cross-linking. IGF-1, insulin, and EGF stimulated [³H]thymidine incorporation by 77, 73, and 87%, respectively. Haft maximal stimulation by IGF-1, insulin, and EGF was produced with 4×10⁻⁹ M, 2.5×10⁻⁸ M, and 8×10⁻¹⁰ M concentrations of these hormones. α-IR3 inhibited stimulation of thymidine incorporation by IGF-1 and insulin but had no effect in EGF-stimulated thymidine incorporation. EGF and high concentrations of insulin both stimulated cell proliferation by 83 and 79%, respectively. These data are consistent with regulation of tubular epithelial proliferation by IGF-1, insulin, and EGF and suggest that the mitogenic activity of both insulin and IGF-1 is mediated by the type 1 IGF receptor. Supported by grants CA37887 and DK32889 from the National Institutes of Health, Bethesda, MD, and by a Medical University of South Carolina institutional grant.     

Separated human breast epithelial and myoepithelial cells have different growth factor requirements in vitro but can reconstitute normal breast lobuloalveolar structure

In order to investigate the specific factors controlling the growth of normal breast cell types, purified populations of human breast epithelial and myoepithelial cells from reduction mammoplasties were grown in primary culture in three defined media and their response to foetal calf serum (FCS), epidermal growth factor (EGF) and basic fibroblast growth factor (FGF2) measured using MTT growth assays. Epithelial and myoepithelial cells differed markedly in their growth requirements. Whereas epithelial cell survival was dependent on the presence of FCS, myoepithelial cell growth was dramatically inhibited by serum. EGF and FGF2 were mitogenic for epithelial cells but not myoepithelial cells, the addition of insulin being the only essential supplement required for myoepithelial cell growth. Heparin inhibited FGF2-stimulated epithelial cell growth but also basal myoepithelial cell proliferation and this inhibition could be overcome by the addition of EGF. Neutralizing antibodies to EGF also inhibited basal myoepithelial cell growth. This suggests the possibility of an autocrine role for a heparin-binding member of the EGF family in the growth of myoepithelial cells. Purified cells combined to form lobuloalveolar structures when incubated in a reconstituted basement membrane matrix (Matrigel) in the presence of EGF and FGF2. J. Cell. Physiol. 171:11–19, 1997. © 1997 Wiley-Liss, Inc.     

Growth control of normal and transformed cells

Both serum factors and protein synthesis are required for normal cell growth. Swiss 3T3 cells require the serum growth factors insulin and EGF (epidermal growth factor) during the initial part of the G₁ period, until they pass a restriction point about 2 h before the initiation of DNA synthesis. Concentration of cycloheximide that inhibit protein synthesis by as much as 70% dramatically lengthen the cell cycle before the restriction point, while the cell cycle after the restriction point remains nearly constant. These results are consistent with a model in which labile proteins are required for transit of cells past the serum-sensitive restriction point. The relation of these findings to the growth control of transformed cells is discussed.     

Raloxifene and desmethylarzoxifene block estrogen-induced malignant transformation of human breast epithelial cells

There is association between exposure to estrogens and the development and progression of hormone-dependent gynecological cancers. Chemical carcinogenesis by catechol estrogens derived from oxidative metabolism is thought to contribute to breast cancer, yet exact mechanisms remain elusive. Malignant transformation was studied in MCF-10A human mammary epithelial cells, since estrogens are not proliferative in this cell line. The human and equine estrogen components of estrogen replacement therapy (ERT) and their catechol metabolites were studied, along with the influence of co-administration of selective estrogen receptor modulators (SERMs), raloxifene and desmethyl-arzoxifene (DMA), and histone deacetylase inhibitors. Transformation was induced by human estrogens, and selectively by the 4-OH catechol metabolite, and to a lesser extent by an equine estrogen metabolite. The observed estrogen-induced upregulation of CYP450 1B1 in estrogen receptor negative MCF-10A cells, was compatible with a causal role for 4-OH catechol estrogens, as was attenuated transformation by CYP450 inhibitors. Estrogen-induced malignant transformation was blocked by SERMs correlating with a reduction in formation of nucleobase catechol estrogen (NCE) adducts and formation of 8-oxo-dG. NCE adducts can be formed consequent to DNA abasic site formation, but NCE adducts were also observed on incubation of estrogen quinones with free nucleotides. These results suggest that NCE adducts may be a biomarker for cellular electrophilic stress, which together with 8-oxo-dG as a biomarker of oxidative stress correlate with malignant transformation induced by estrogen oxidative metabolites. The observed attenuation of transformation by SERMs correlated with these biomarkers and may also be of clinical significance in breast cancer chemoprevention.     

Serum-induced chromosome damage and neoplastic transformation of mouse cells in vitro

Summary In previous studies, mouse cells grown in medium supplemented with horse serum (HS) developed more chromosomal aberrations and underwent malignant transformation earlier than cells from the same pool grown with fetal bovine serum (FBS) supplement. In the present study cells derived from C3H_f/HeN mouse embryos were grown in medium NCTC-135 supplemented with various combinations of large- and small-molecule fractions of HS and FBS in an effort to determine the effective components. The results indicate that the large-molecule fraction of HS (mare or stallion) produces alterations in chromosome number and structure. HS is also shown to cause chromatid breaks and exchanges at or near the centromere in contrast to fluorescent-light-induced breaks which occur randomly along the chromatid. However, efforts to control completely chromosome stability and malignant transformation through the use of large-and small-molecule fractions of HS and FBS or combinations thereof were unsuccessful. In comnection with this study, diagnosis of malignant transformation in vitro was made by a direct sampling method based on cytologic criteria previously described and documented. With one exception, the diagnoses of 11 different cell lines were consistent with results of in vivo assays.     

2-DE analysis of a new human cell line EM-G3 derived from breast cancer progenitor cells and comparison with normal mammary epithelial cells

We performed a 2-DE analysis of proteins of the newly established spontaneously immortalized clonal cell line EM-G3 derived from a primary lesion of infiltrating ductal breast carcinoma. EM-G3 cells may represent progenitors of the mammary epithelial cells spontaneously immortalized in early phase of cancerogenesis. We compared the protein profile of EM-G3 line with proteins from populations of normal mammary epithelial cells (NME), and determined the phenotype of both types of cells. NME cells are a mixture of both main cell types in breast epithelia, myoepithelial and luminal cells. The EM-G3 breast cancer cell line has a unique basal-like phenotype. We identified proteins that are differently expressed in these cells. Cytokeratin 16, cytokeratin 19, squamous cell carcinoma antigen 1, caphepsin B and caspase 14 were predominantly expressed by NME cells. Cytokeratin 13, isoelectric variant of annexin 5, isoelectric variant of chloride intracellular channel protein 1, glyoxalase 1 and glutamine synthetase were predominantly expressed by EM-G3 cells. The proteins up-regulated in EM-G3 cells may represent potential protein markers of mammary epithelial cells progenitors and may be important in early phase of carcinogenesis.     

Rat pituitary tumor cells in serum-free culture. II. Serum factor and thyroid hormone requirements for estrogen-responsive growth

Summary The effect of 17β-estradiol (E₂) on growth of GH₄C₁ rat pituitary tumor cells was investigated under serum-free conditions and with medium containing charcoal-extracted serum. Serum-free TRM-1 medium was a 1∶1 (vol/vol) mixture of F12-DME supplemented with 50 μg/ml gentamicin, 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 μg/ml insulin, 10 μg/ml transferrin, 10 ng/ml selenous acid, 10 nM 3,5,3′-triiodothyronine (T₃), 50 μM ethanolamine, and 500 μg/ml bovine serum albumin. The cells grew continuously in TRM-1 but were E₂ responsive only when growth was retarded by reducing the T₃ concentration to 10 pM (TRM-MOD). Addition of 1 to 10 nM E₂ to TRM-MOD increased growth by 0.3 to 0.9 cell population doublings over controls in 9 d. By using medium supplemented with charcoal-extracted sera, basal growth became 1 to 1.5 cell population doublings in 9 d. Addition of 0.1 pM E₂ to medium containing charcoal-extracted serum caused a significant increase in cell number whereas pM-nM concentrations stimulated 200 to 570% increases over controls. The effect of steroid hormone was the same in phenol-red-containing and indicator-free medium. The data presented confirm that the major requirements for demonstration of estrogenic effects in culture were optimum concentrations of thyroid hormones and the presence of yet-to-be-characterized serum factors. This work was supported by National Cancer Institute (Bethesda, MD) grants CA-26617 and CA-38024, American Cancer Society grant BC-255, and grant 2225 from The Council for Tobacco Research, U.S.A., Inc.