Quantitation of human mammary epithelial antigens in cells cultured from normal and cancerous breast tissues

Summary A sensitive radioimmunoassay technique was developed to quantitatite the level of human breast celltype specific antigens on cells from normal breast and from various established cell lines of breast and nonbreast origins. Polyacrylamide gel electrophoresis revealed four major proteinaceous components (150,000; 75,000; 60,000; and 48,000) in human milk fat globule membranes that were used to immunize rabbits in order to elicit antimammary epithelial cell antibody. Antisera obtained were rendered specific by abosorptions and were able to recognize three specific mammary epithelial components of the breast epithelial cell. Human mammary epithelial (HME) antigen expression was highest (1290 ng/10⁶ cells) in normal breast epithelial cells from primary cultures of normal breasts. Lower levels (range: 955 to 330 ng/10⁶ cells) were found in breast epithelial cells from cell lines established from cancerous breast tissue. Cells of nonbreast origins as well as fibroblasts from breast gave much lower values (less than 30 ng/10⁶ cells). On treatment, with trypsin, of two breast epithelial cell lines (MDA-MB-157 and MCF-7) 80 to 85% of their HME antigen expression was lost, suggesting that a majority of these breast antigens reside on the cell surface. This work was Supported by Grant PTD-99 from the American Cancer Society, Grant CA19455 and CA20286 from the National Cancer Institute, and Biomedical Research Support Grant RR05467 from the National Institutes of Health. Most cells used in the present study were produced with support from National Cancer Institute Contract Y01-CP8-0500, Biological Carcinogenesis Branch, Division of Cancer Cause and Prevention, under the auspices of the Office of Naval Research and the Regents of the University of California.     

Histochemical identification of cultured cells from human endometrium

Summary Histochemical techniques have been applied to the identification of cell types cultured from human endometrium. Previous work from this laboratory characterized two principtal cell types found in cultures of endometrium: a mature epithelial cell and another cell which was classified as the endometrial stromal cell based on light and electron microscopy. In this report we compare the histochemical staining of endometrial tissue in frozen sections to that of cultured cells. These results confirm the epithelial and stromal nature of the respective cell types. Several markers were found that could distinguish between cells of epithelial and stromal origin. The enzymes alkaline phosphatase, γ-glutamyltranspeptidase, peroxidase, and β-glucuronidase were localized in glandular and surface epithelia in frozen sections and in colonies of epithelial cells in culture. Stroma in frozen sections and cultured stromal cells contained leucine aminopeptidase and fibronectin. Epithelial sections and in culture could also be distinguished from cells of stromal origin by preferential binding of lotus and peanut lectin. Several other markers were found in both endometrial epithelium and stroma. J. M. S. was recipient of National Research Service Award CA09156 (National Cancer Institute); K. G. N. was recipient of National Research Service Award ES07017 (National Institute of Environmental Health Sciences); and D. G. K. was recipient of Research Career Development Award CA00431 from the National Cancer Institute, Bethesda, MD. Supported by Grant CA 31733 from the National Cancer Institute, Bethesda, MD.     

Development of two cloned epithelial cell lines from normal adult mouse and rat ventral prostates

Summary Two epithelial cell lines were established, one from adult C3H mouse and one from adult Fischer rat ventral prostate. These cell lines were obtained from explant cultures, using Ham's F12 medium supplemented with HEPES, insulin, testosterone, hydrocortisone, epidermal growth factor, and 7.5% fetal bovine serum. A low concentration of trypsin and EDTA in Ca⁺⁺-and Mg⁺⁺-free phosphate buffer was used for passaging the cells. The rat cell line was established following implantation of prostate tissue in nude mice. These cell lines stained positively for acid phosphatase and were dependent upon epidermal growth factor for growth. Morphological studies, including electron microscopy, revealed a highly characteristic epithelial morphology of both cell lines. These cell lines have hypotetraploid chromosome numbers and are capable of metabolizing benzo(a)pyrene. We propose the application of these cells as models for the study of prostate carcinogenesis. This work was supported in part by Grant CA-21, 746, and by the Electron Microscope Core Facility on Grant CA-14,089, from the National Cancer Institute, National Institutes of Health, Bethesda, MD.     

Effect ofd-valine and cytosine arabinoside on [3H]thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

Summary Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by [³H]thymidine uptake, is very low. Inl-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured ind-valine medium was significantly lower than that of cells cultured inl-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured ind-valine medium was significantly higher than that of cells cultured inl-valine medium. Cytosine arabinoside decreased the number of labeled cells in bothl-valine andd-valine cultures. From these results, it appears thatd-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures. This research was sponsored by grants from the National Institute of Child Health and Human Development, Bethesda, MD (HD-03820, HD-11816, HD-05797), and the Mellon Foundation.     

Culture of adult rat lung cells: Benzo(a)pyrene metabolism and mutagenesis

Summary A method is described for obtaining and culturing large numbers of lung cells from normal adult male rats. The lungs were perfused in situ to remove blood cells and then perfused via the trachea with a trypsin-collagenase solution to initiate tissue digestion. The tissue was further digested in the enzyme solution and approximately 2×10⁸ viable lung cells were obtained per animal. Primary cultures contained a mixed cell population. Through eight subcultures about 70% of the cell population possessed an epithelial-like morphology, whereas the remaining 30% was fibroblast-like. Three clones of epithelial-like cells were isolated at the fourth subculture. The mass culture lung cells and the epithelial-like clone that was studied retained a normal karyotype and did not grow in soft agar. Both the mass culture cells and the epithelial clone metabolized the lung carcinogen benzo(a)pyrene (BP) to water-soluble products. Furthermore, the mass culture lung cells metabolized BP to intermediate(s) which mutated Chinese hamster V79 cells from ouabain sensitivity to ouabain resistance. These lung cell cultures have potential use in cell transformation, mutation and carcinogen metabolism studies. Visiting scientist from Hungary. This research was supported by Grant 5 R01 CA20022 and Public Health Service Contract N01 CP33278 from the Division of Cancer Cause and Prevention, National Cancer Institute, National Institutes of Health.     

Junctional communication pattern of cells cultured from human milk

Junctional communication between cultured human milk cells and fibroblasts derived from human foreskin or mammary gland has been investigated. The dividing cells in primary cultures from milk, which have been shown to be epithelial by immunological and morphological criteria, showed selectivity in communication and did not transfer ³H-labelled nucleotides to either type of fibroblast. With passage the communication phenotype changed, selectivity was lost and a morphologically homogeneous cell type emerged which communicated not only with both types of fibroblast, but also with the milk epithelial cells found in primary culture.     

Aminocentesis cells: Culture,cryogenic storage,isozymes, and finite life in vitro

Summary Forty-six cell cultures established from amniocentesis fluids were preserved in liquid nitrogen and later recovered from the frozen state with little loss of viability as compared to prefreeze viability. Five to 10% glycerol was found to be optimal for preservation in liquid nitrogen, and as few as 5×10⁵ viable cells per frozen ampule could initiate cell growth. Storage in liquid nitrogen did not affect the genetic stability of glucose-6-phosphate dehydrogenase, lactate dehydrogenase, malic dehydrogenase, leucine aminopeptidase, acid phosphatase, or 6-phosphogluconic acid dehydrogenase isozymes of the amnion cultures. These studies were supported by Contract NIH-NIGMS-72-2070, Grant CA-04953-13 from the National Cancer Institute; General Research Support Grant FR-5582 from the National Institutes of Health; and Grant-in-Aid Contract M-43 from the State of New Jersey. Recipient of Research Career Award 5-K3,16, 749 from the National Institutes of Health.     

Ciliated cells in vitamin A-deprived cultured hamster tracheal epithelium do divide

Summary The pseudostratified tracheal epithelium, composed of a heterogeneous phenotypically varying cell population, was studied with respect to the in vitro cell proliferative activity of differentiated epithelial cells. Ciliated tracheal epithelial cells so far have been considered to be terminally differentiated, nonproliferating cells. Tracheal organ cultures obtained from vitamin A-deprived Syrian Golden hamsters were cultured in a vitamin A-deficient, serum-free, hormone-supplemented medium. In vitamin A-deprived tracheal epithelium treated with physiologically active all-trans retinol and low cigarette-smoke condensate concentrations it is possible to stimulate the cell proliferation of both basal and columnar cells. Therefore, the probability of finding proliferating columnar cells was increased compared with the in vivo and the vitamin A-deprived situation in which cell proliferative activity is relatively low. In the presence of cigarette-smoke condensate in a noncytotoxic concentration, basal, small mucous granule, ciliated, and indifferent tracheal epithelial cells incorporated [methyl-³H]-thymidine into the DNA during the S phase. The finding that ciliated cells were labeled was supported by serial sections showing the same labeled ciliated cell in two section planes separated by 2 to 3 μm, without labeled epithelial cells next to the ciliated cell. Furthermore, a ciliated tracheal epithelial cell incorporating [methyl-³H]thymidine into DNA was also seen in tracheal cultures of vitamin A-deprived hamsters treated with all-trans retinol in a physiologic concentration. The present study was financially supported by the Scientific Advisory Committee on Smoking and Health (Dutch Cigarette Industry Foundation) and the Ministry of Welfare, Health and Cutural Affairs.     

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)¹. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium². Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production^3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras⁶ and modified by Kopan et al.⁷, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies⁸. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses⁹. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.     

A method for the rapid establishment of normal adult mammalian colonic epithelial cell cultures

Summary Normal colonic epithelial cell cultures of mammalian origin are required to facilitate the study of both normal cellular functions as well as pathogenesis of certain (human) colonic diseases. To date, little information is available regarding the growth requirements of colonic epithelial cells in culture of eitehr animal or human origin. Such data would enable the development of a long-term culture system for these cells. In this study, we present methodology that results in the establishment of homogeneous cultures of adult rabbit colonic epithelial reproducibly, quickly, and in quantity. The epithelial nature of the cultures is unambiguously established by intermediate filament typing using antikeratin antibodies. Such culutres can now be used for a variety of functional studies as well as to investigate the growth requirements of colonic epithelial in culture. This work was supported by the Blinder Foundation for Crohn’s Disease Research, Harbor UCLA IBD Center (AM 36200) and grant AM 27806 from the National Institutes of Health, Bethesda, MD.     

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells^1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells³. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)^4-5. Sufficient numbers of cells can be obtained from one individual''s tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation^4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic^5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.     

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.     

Primary cultures of normal rat kidney proximal tubule epithelial cells for studies of renal cell injury

Summary Normal rat kidney proximal tubule epithelial cell cultures were obtained by collagenase digestion of cortex and studied for 10 days. To assess the purity of the seeding suspension, we histochemically demonstrated γ-glutamyltranspeptidase in >95% of the starting material. To identify cell types in cultures, we investigated several markers. Cells stained positively for lectinArachis hypogaea (rat proximal tubule) and negatively forLotus tetragonolobus (rat distal tubule). Intermediate filament expression of cytokeratin confirmed the epithelial differentiation of the cultured cells. Using indirect immunofluorescence, we found that cultures were negative for vimentin and Factor VIII. Cells exhibited activities of two brush border enzymes, γ-glutamyltranspeptidase and leucine aminopeptidase, and Na⁺-dependent glucose transport activity. Multicellular domes were evident in the Week 2 of culture. Proliferation was studied by comparing growth factor-supplemented serum-free medium to cells grown in serum; growth enhancers included insulin, hydrocortisone, transferrin, glucose, bovine albumin, and epidermal growth factor. Cells proliferate best in medium with 5 or 10% serum and in serum-free medium supplemented with insulin, hydrocortisone, transferrin, glucose, and bovine albumin. Proliferation was assessed by determining cell number (population doublings). By light microscopy, the cells were squamous with numerous mitochondria, a central nucleus, and a rather well-defined homogeneous ectoplasm. By electron microscopy, the cells were polarized with microvilli and cell junctions at the upper surface and a thin basal lamina toward the culture dish. These data show that the proximal tubule epithelial cells retain a number of functional characteristics and that they represent an excellent model for studies of normal and abnormal biology of the renal proximal tubule epithelium. This project was supported by grant 2-R01-DK15440-16A1 from the National Institutes of Health, Bethesda, MD, and by grant N0001 4-88-K-0427 from the Department of the Navy, Washington, DC.     

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors^1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension^3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture³. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized⁴. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells⁵. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals⁶. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors⁷.Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion^8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)^16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections²³. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.     

Monolayer culture of human endometrium: Methods of culture and identification of cell types

Summary Monolayer cultures can be established from human endometrial tissue after enzymatic dispersal into isolated glands or single cells. Three cell types that have distinct morphology by light and electron microscopy are observed in the resulting primary cultures. One cell type, an elongated spindle cell, is similar in appearance to fibroblasts derived from other tissues. A second cell type forms colonies of tightly cohesive cells, ranging in shape from oval to polygonal. These cells have typical organelles and junctional complexes characteristic of epithelial cells from the endometrium. The third cell type assumes a pavement-like appearance composed of polygonal cells when viewed by phase contrast microscopy, but lacks distinctive ultrastructural features of epithelial cells. These cells in culture resemble the endometrial stromal cell, the predominant cell type of the human endometrium in vivo. The epithelial cell does not survive subculturing but the other two cell types can be passaged through several generations and can be stored in liquid nitrogen and subsequently returned to culture. This work was supported by contract N01-CP75956 and grant R01-CA31733 from the National Cancer Institute. V. A. Varma is a recipient of an American Cancer Society fellowship; B. H. Dorman, a predoctoral fellowship from the Chemical Industry Institute of Toxicology; J. M. Siegfried, a training grant (CA09156) from the National Cancer Institute; and D. G. Kaufman, a Research Career Development Award (K04-CA-00431) from the National Cancer Institute.     

Human oral epithelial cell culture II. Keratin expression in fetal and adult gingival cells

Summary Epithelial cells from human fetal and adult gingiva were cultured in keratinocyte growth medium (KGM), a serum-free medium. The expression of keratin proteins in these cells was evaluated using immunohistochemistry and SDS-PAGE-immunoblot analysis and compared with expression in the tissue. Keratins 5, 6, 14, 16, and 19 were identified in cells cultured from both fetal and adult tissues. K19 was localized in basal cells of fetal oral tissue but was not seen in adult gingiva (except for scattered Merkel cells). K1 and K10 were expressed in tissue, but not in cultured cells. The keratin profiles of cultured epithelial cells from several adult donors were similar and were identical in cultures from primary through Passage 5. K13, a differentiation-specific keratin, was expressed in all suprabasal cells of fetal oral epithelium, but shows only spotty expression in adult gingival tissue. K13 was expressed in cultures of fetal cells, but very weakly or not at all in cultures of adult cells. K13 expression was greater in cultures grown with physiologic calcium concentrations (1.2 mM) than in those grown at 0.15 mM or less. Our findings are consistent with basal-like characters of these cells in 0.15 mM calcium growth conditions. Differentiation of fetal oral cells in culture to the suprabasal basal cell stage in 1.2 mM Ca²⁺ is shown by the expressionof K13. This work was supported by Biomedical Research grant RR05346, National Institutes of Health grant DE04660, University of Washington Graduate Fund and Hack Foundation Fund, Department of Periodontology, University of Washington.     

Development and limitations of lentivirus vectors as tools for tracking differentiation in prostate epithelial cells

To investigate hierarchy in human prostate epithelial cells, we generated recombinant lentiviruses, infected primary cultures and cell lines, and followed their fate in vitro. The lentiviruses combined constitutive promoters including CMV and β-actin, or late-stage differentiation promoters including PSCA (prostate stem cell antigen) and PSAPb (prostate specific antigen/probasin) driving expression of monomeric, dimeric and tetrameric fluorescent proteins. Significantly, rare CD133⁺ cells from primary prostate epithelial cultures were successfully infected and activation of late-stage promoters was observed in basal epithelial cultures following induction of differentiation. Lentiviruses also infected CD133⁺ cells within the P4E6 cell line. However, promoter silencing was observed in several cell lines (P4E6, BPH-1, PC3). We examined the promoter methylation status of the lentiviral insertions in heterogeneously fluorescent cultures from PC3 clones and found that DNA methylation was not the primary mechanism of silencing of the CMV promoter. We also describe limitations to the lentivirus system including technical challenges due to low titers and low infection efficiency in primary cultures. However, we have identified a functional late-stage promoter that indicates differentiation from a basal to a luminal phenotype and demonstrate that this strategy for lineage tracking of prostate epithelial cells is valid with further optimisation.     

Ultrastructure of human endometrial epithelium in monolayer culture with and without steroid hormones

Summary Colonies of cells of epithelioid appearance were identified in monolayer cultures grown up to 50 days from normal human endometrial cell suspensions obtained by a method designed to insure a maximum harvest of glandular cells. Groups of these cells were separated from stromal cells by means of cloning cylinders. Studies comparing the ultrastructure of cells of this type to fresh endometrial tissue revealed a number of similarities. The morphological characteristics common to both types of samples included junctional complexes, perinuclear microfilaments and microvilli with glycocalyx. Other common features were prominent nucleoli, well developed Golgi, rough endoplasmic reticulum and membranebound electron-dense bodies in the cytoplasm. A stripping technique applie to the fetal bovine serum used in the nutrient medium made it possible to initiate cultures in a steroidfree environment and to maintain them in the presence of the specified concentration of estradiol and/or progesterone. Isolation of epithelial cells of endometrium in monolayer culture may provide a useful model system in which to study the specific effects of steroid hormones on cellular function and differentiation. Supported by grants from the National Institutes of Health (CA 18678 and CA 07368).     

Expression of Betapapillomavirus Oncogenes Increases the Number of Keratinocytes with Stem Cell-Like Properties

Human papillomaviruses (HPV) of genus Betapapillomavirus (betaPV) are associated with nonmelanoma skin cancer development in epidermodysplasia verruciformis (EV) and immunosuppressed patients. Epidemiological and molecular studies suggest a carcinogenic activity of betaPV during early stages of cancer development. Since viral oncoproteins delay and perturb keratinocyte differentiation, they may have the capacity to either retain or confer a “stem cell-like” state on oncogene-expressing cells. The aim of this study was to determine (i) whether betaPV alters the expression of cell surface markers, such as CD44 and epithelial cell adhesion molecule (EpCAM), that have been associated with epithelial stemness, and (ii) whether this confers functional stem cell-like properties to human cutaneous keratinocytes. Fluorescence-activated cell sorter (FACS) analysis revealed an increase in the number of cells with high CD44 and EpCAM expression in keratinocyte cultures expressing HPV type 8 (HPV8) oncogenes E2, E6, and E7. Particularly through E7 expression, a distinct increase in clonogenicity and in the formation and size of tumor spheres was observed, accompanied by reduction of the epithelial differentiation marker Calgranulin B. These stem cell-like properties could be attributed to the pool of CD44^high EpCAM^high cells, which was increased within the E7 cultures of HPV5, -8, and -20. Enhanced EpCAM levels were present in organotypic skin cultures of primary keratinocytes expressing E7 of the oncogenic HPV types HPV5, -8, and -16 and in clinical samples from EV patients. In conclusion, our data show that betaPV may increase the number of stem cell-like cells present during early carcinogenesis and thus enable the persistence and accumulation of DNA damage necessary to generate malignant stem cells.     

Comparative C19-radiosteroid metabolism by MA 160 and HeLa cell lines

Summary Since the designation of the human MA 160 line as prostatic epithelial cells has been questioned and the possibility of HeLa cross contamination raised, this comparative study of C₁₉-radiosteroid transformation in MA 160 and HeLa monolayer cultures was done to determine whether these cells possess the distinguishing features of reductive and oxidative androgen metabolism expected in male and female genital organs, respectively. We compared the radiometabolite patterns produced by incubating [¹⁴C]testosterone (300nM) and [³H]testosterone (3nm) and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) with cultures of prostatic MA 160 and HeLa Parent, TCRC-1, TCRC-2 and ATC 229 cells. C₁₉-Radiosteroid metabolite patterns from MA 160 cell incubations also were compared with patterns generated by MA 196 fibroblasts from abdomnal skin of the same donor. MA 160 cells metabolized radiotestosterone predominantly to 5α-dihydrotestosterone, 5α-androstane-3α,17β-diol and 5α-androstane-3β,17β-diol. The diol epimers were the principal metabolites of 5α-dihydrotestosterone radiosubstrate. In contrast, radiotestosterone metabolism by MA 196 and HeLa Parent, TCRC-1 and TCRC-2 cells was overwhelmingly to the 17-oxosteroids 4-androstene-3,17-dione and androsterone. Another pathway was operative in HeLa 229 and, to a minor extent, in TCRC-1, which converted radiotestosterone to 4-androstene-3α,17β-diol and 5α-androstane-3α,17β-dol, with little formation of 5α-dihydrotestosterone. MA 160 cells thus metabolize radiotestosterone preponderantly to 5α-reduced 17β-hydroxysteroids as expected for prostatic epithelial cells, whereas HeLa cells show heterogeneity in metabolizing the labeled hormone by the alternative 17-oxosteroid and Δ⁴ pathways. This work was supported by Public Health Service Research Grants CA 13417 and CA 12924 from the National Cancer Institute, AM 11011 from the National Institute of Arthritis, Metabolism and Digestive Diseases, and by appropriations of the Commonwealth of Massachusetts, Item No. 4532-9003-01.