In vitro deregulation of markers characteristic of human prostate epithelial cells

We have screened primary cultures of human prostate for the expression of markers reported to be characteristic of specific cell lineages in vivo, in order to ascertain whether human prostate cells in vitro maintain and reflect their in vivo differentiated phenotypes and to evaluate the homogeneity of the populations of cells that can be derived from this tissue. Using single and dual stain immunofluorescent microscopy to analyse very early organoid and subsequently derived monolayer stage cultures, we have observed that expression of markers characteristic of human prostate epithelial cells in vivo is deregulated within 48h, indicating that dissociation of human prostate tissue and cultivation of prostate epithelial cells in culture can result in promiscuous expression of cell type specific markers of prostate epithelial cells. These observations have important implications for studies of cell lineage and differentiation of prostate cells in vitro.     

Immortalization and malignant transformation of eukaryotic cells

The process of cellular transformation has been amply studied in vitro using immortalized cell lines. Immortalized cells never have the normal diploid karyotype, nevertheless, they cannot grow over one another in cell culture (contact inhibition), do not form colonies in soft agar (anchorage-dependent growth) and do not form tumors when injected into immunodeficient rodents. All these characteristics can be obtained with additional chromosome changes. Multiple genetic rearrangements, including whole chromosome and gene copy number gains and losses, chromosome translocations, gene mutations are necessary for establishing the malignant cell phenotype. Most of the experiments detecting transforming ability of genes overexpressed and/or mutated in tumors (oncogenes) were performed using mouse embryonic fibroblasts (MEFs), NIH3T3 mouse fibroblast cell line, human embryonic kidney 293 cell line (HEK293), and human mammary epithelial cell lines (mainly HMECs and MC-F10A). These cell lines have abnormal karyotypes and are prone to progress to malignantly transformed cells. This review is aimed at understanding the mechanisms of cell immortalization by different "immortalizing agents", oncogene-induced cell transformation of immortalized cells and moderate response of the advanced tumors to anticancer therapy in the light of tumor "oncogene and chromosome addiction", intra-/intertumor heterogeneity, and chromosome instability.     

Immortalized cells as experimental models to study cancer

The development of cancer is a multi-step process in which normal cells sustain a series of genetic alterations that together program the malignant phenotype. Much of our knowledge of cancer biology results from the detailed study of specimens and cell lines derived from patient tumors. While these approaches continue to yield critical information regarding the identity, number, and types of alterations found in human tumors, further progress in understanding the molecular basis of malignant transformation depends upon the generation and use of increasingly sophisticated experimental models of cancer. Over the past several years, the recognition that telomeres and telomerase play essential roles in regulating cell lifespan now permits the development of new models of human cancer. Here we review recent progress in the use of immortalized human cells as a foundation for understanding the molecular basis of cancer.     

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening.     

Establishment of a three-dimensional human prostate organoid coculture under microgravity-simulated conditions: Evaluation of androgen-induced growth and psa expression

Summary A novel in vitro human prostate cancer model was established by using a coculture technique in which isolated human prostate fibroblasts were observed to grow as a mixed culture with isolated human prostate cancer cells (LNCaP) on microcarrier beads under microgravity-simulated conditions. This model appears to be promising and deserves further exploration because: (a) cocultured human prostate fibroblasts and cancer epithelial cells appear to undergo patterns of histogenesis similar to those observed in human prostate tumors and (b) unlike the conventional cell culture on plastic dishes, cocultured human prostate fibroblasts and LNCaP cells in microgravity-simulated conditions responded to the inductive signals of growth and differentiation from dihydrotestosterone in a manner similar to that observed in the in vivo condition. These results offer an opportunity to examine molecular mechanisms of cellular signaling in response to androgen stimulation during normal and aberrant human prostate development. The microgravity-simulated three-dimensional prostate epithelial cell culture with prostate fibroblasts can be further explored as an ideal in vitro model for the study of normal and neoplastic prostate development. This model could also be adopted as a drug screening program for the discovery of novel therapeutic agents in the treatment of human prostate cancer and benign hyperplastic growth.     

A method for prolonged survival of primary cell lines

Summary We have established a means for prolonged survival of primary cell cultures and establishment of continuous cell lines without genetic manipulations. Primary cultures of granulosa cells degenerate rapidly in vitro by a spontaneous onset of apoptotic cell death. Earlier attempts to circumvent this limitation have included transformation with oncogenes, spontaneous immortalization of primary cultures, and chemical carcinogenesis. We have found that addition of a complex of growth-promoting compounds, carrier proteins, and factors isolated from porcine follicular fluid to standard culture medium allows, reproducibly, the establishment of continuous porcine primary granulosa cell lines with genetic stability. This same supplement allows the prolonged survival of primary cell cultures derived from adult rat ovaries. The rat ovary primary cultures consisted of mixed phenotypes, including epithelial, neuron-like, and mesenchymal cell types. Numerous cells stain positive for alkaline phosphatase in these cultures. Other primary cell lines were established from embryonic rat liver and from adult rat lungs, using the same supplement. The survival effect is reversible because cells degenerate when the supplement is removed. Therefore, the cell lines have neither acquired properties of a tumor cell line nor have they been immortalized by a virus infection. We expect that our approach will open the door to prolonged survival of other primary cell types.     

Evaluation of the chemopreventive potential of retinoids using a novel in vitro human prostate carcinogenesis model

The prevalence of prostatic intraepithelial neoplasia (PIN) and latent prostatic carcinoma, representing multiple steps in carcinogenesis and progression to invasive carcinoma, makes them relevant targets for prevention. A unique family of human prostate epithelial cell lines, which mimic steps in prostate carcinogenesis and progression, were used to evaluate the chemopreventive potential of all-trans-retinoic acid (RA) and N-(4-hydroxyphenyl)retinamide (4-HPR). The effects of RA and 4-HPR on anchorage-dependent growth of an immortalized, non-tumorigenic cell line RWPE-1 and two tumorigenic cell lines, WPE1-NB14 and WPE1-NB11, derived from RWPE-1 by exposure to N-methyl-N-nitrosourea (MNU), were examined. Both tumorigenic cell lines grow more rapidly than the parent RWPE-1 cell line in monolayer culture. Further, while RWPE-1 cells do not form colonies in agar, both tumorigenic cell lines do, with a colony forming efficiency (CFE) of 1.85 and 2.04% for WPE1-NB14 and WPE1-NB11 cells, respectively. Both RA and 4-HPR inhibited anchorage-dependent growth of all cell lines and anchorage-independent growth of WPE1-NB14 and WPE1-NB11 cells, in a dose-dependent manner, however, 10 times more RA than 4-HPR was required to produce the same effect. RWPE-1 cells are not invasive but WPE1-NB11 cells are significantly more invasive than WPE1-NB14 cells. Both RA and 4-HPR inhibited invasion in vitro by WPE1-NB11 and WPE1-NB14 cells where the more malignant WPE1-NB11 cells showed greater inhibition of invasion by 4-HPR than by RA. Overall, 4-HPR was more effective than RA in inhibiting growth and invasion but the response varied amongst the cell lines. These three cell lines mimic progressive steps in carcinogenesis and progression, from immortalized, non-tumorigenic RWPE-1 cells, to the less malignant WPE1-NB14 to the more malignant WPE1-NB11 cells, and provide powerful models for studies on secondary and tertiary prevention, i.e. promotion and progression stages, respectively, of prostate cancer.     

Immortalization and malignant transformation of Eukaryotic cells

The process of cellular transformation has been amply studied in vitro using immortalized cell lines. Immortalized cells never have the normal diploid karyotype, nevertheless, they cannot grow over one another in cell culture (contact inhibition), do not form colonies in soft agar (anchorage-dependent growth) and do not form tumors when injected into immunodeficient rodents. All these characteristics can be obtained with additional chromosome changes. Multiple genetic rearrangements, including whole chromosome and gene copy number gains and losses, chromosome translocations, gene mutations are necessary for establishing the malignant cell phenotype. Most of the experiments detecting transforming ability of genes overexpressed and/or mutated in tumors (oncogenes) were performed using mouse embryonic fibroblasts (MEFs), NIH3T3 mouse fibroblast cell line, human embryonic kidney 293 cell line (HEK293), and human mammary epithelial cell lines (mainly HMECs and MCF10A). These cell lines have abnormal karyotypes and are prone to progress to malignantly transformed cells. This review is aimed at understanding the mechanisms of cell immortalization by different “immortalizing agents”, oncogene-induced cell transformation of immortalized cells and moderate response of the advanced tumors to anticancer therapy in the light of tumor “oncogene and chromosome addiction”, intra-/intertumor heterogeneity, and chromosome instability.     

Development and characterization of breast carcinoma cell lines as in vitro and in vivo models for breast cancer diagnosis and therapy

Summary To establish a model system for preclinical radioimmunotherapy studies, attempts were made to graft 16 different human breast carcinoma cell lines into BALB/c nu/nu (nude) mice. Nine produced serially transplantable tumors growing at a variable rate, whereas seven failed to do so. Conversely, three new cell lines were established in monolayer culture from transplantable human breast tumors in nude mice. Twelve selected tumors and their corresponding cell lines were characterized for DNA ploidy, % S-phase, and breast epithelial mucin expression by immunohistochemistry and flow cytometry. A wide diversity of these cellular characteristics were found in that each tumor was unique and distinct from the others. DNA ploidy differed among the tumors but was not affected by switching between in vitro to in vivo growth. Some tumors expressed similar levels of the breast mucin both in vitro and in vivo, whereas most expressed lower levels as transplantable tumors. There was a good correlation between immunohistochemical and flow cytometric determination of surface and cytoplasmic mucin expression, and with both techniques estrogen and progesterone receptor positive tumors had significantly higher levels of mucin expression than receptor negative tumors. These 12 transplantable breast tumors, with their corresponding cell lines, provide an excellent model system for testing radioimmunotherapy and other therapeutic reagents because they exhibit diverse phenotypic characteristics that represented a mini-population of breast cancer patients’ tumors, allowing assessment of the effect of therapy when confronted with different breast tumors’ genotype and phenotype.     

Development and characterization of new immortalized human breast cell lines

New human breast cell lines were developed from metastatic breast cancer tissues and normal breast tissues. Primary cultures were initiated from cellular outgrowths of explanted tissues or from mechanically isolated cells in two serum-free media. Cell cultures derived from both cancer and normal tissues were immortalized with pRSV-T plasmid to generate permanent breast cell lines that exhibited an epithelial morphology. Cell lines generated in this study were characterized with respect to morphology, growth rate, karyotype, presence of specific genes, and the expression of epithelial and breast markers. The cell lines expressed the epithelial cell markers, cytokeratins 8 and 18, and retained the capacity to produce human milk fat globulin. They also express the BRCA-1, erbB2, and EGF receptor genes and possess the H-ras, K-ras, and p53 genes. Preliminary data showed that one of the new cancer cell lines was highly sensitive to the cytotoxic action of taxol. It is envisioned that the new breast cell lines will be useful as targets for identification of therapeutic agents against breast cancer and as models for carcinogenesis studies. This revised version was published online in June 2006 with corrections to the Cover Date.     

Derivation and characterization of putative pluripotential embryonic stem cells from preimplantation rabbit embryos

We have derived putative embryonic stem (ES) cell lines from preimplantation rabbit embryos and report here their initial characterization. Two principal cell types emerged following serial passage of explanted embryos, and each has subsequently given rise to immortalized cell lines. One cell type has morphology identical to primary outgrowths of trophectoderm, is strictly feeder-cell dependent, and spontaneously forms trophectodermal vesicles at high cell density. The second type appears to represent pluripotent ES cells derived from the inner cell mass as evidenced by (1) ability to grow in an undifferentiated state on feeder layers, (2) maintenance of a predominantly normal karyotype through serial passage (over 1 year), and (3) ability to form embryoid bodies, which form terminally differentiated cell types representative of ectoderm, mesoderm, and endoderm. These ES cells may ultimately be suitable for introduction of germline mutations (via homologous recombination). The rabbit's size, reproductive capability, and well-characterized physiology make it suitable for a wide range of investigations, particularly for development of large animal models of human disease. © 1993 Wiley-Liss, Inc.     

Production of IGF-II-related peptide by an anaplastic cells line (AT-3) established from the dunning prostatic carcinoma of rats

Summary AT-3 cells, one of anaplastic cell lines established from the Dunning prostatic carcinoma of rats, were able to grow under serum-free conditions in a state of suspension detached from a substratum. Radioimmunoassays using monoclonal antibody against rat insulin-like growth factor II (IGF-II) revealed the presence of IGF-II-related peptide in acid-ethanol extracts extracsts of lyophilized serum-free media conditioned by AT-3 cell. The peptide contents in the culture media increased with increase in cell number; 71 ng at 3.0 × 10⁶ cells and 449 ng at 4.6 × 10⁷ cells. IGF-II-related peptide was hardly detectable in acid-ethanol extracts of AT-3 cells harvested after 13-days culture. These results indicate that AT-3 cells produce IGF-II-related peptide ana may release it into the culture media. Editor's statement One or more members of the insulin-like growth factor family have been established previously as mitogen for isolated prostate cells. This report suggests that IGF-II member of the family may be involved in autocrine support of cells from highly malignant prostate tumors.     

A conditionally immortalized cell line model for the study of human prostatic epithelial cell differentiation

In the normal human prostate, undifferentiated proliferative cells reside in the basal layer and give rise to luminal secretory cells. There are, however, few epithelial cell lines that have a basal cell phenotype and are able to differentiate. We set out to develop a cell line with these characteristics that would be suitable for the study of the early stages of prostate epithelial cell differentiation. We produced a matched pair of conditionally immortalized prostate epithelial and stromal cell lines derived from the same patient. The growth of these cells is temperature dependent and differentiation can be induced following a rise in culture temperature. Three-dimensional co-cultures of these cell lines elicited gland-like structures reminiscent of prostatic acini. cDNA microarray analysis of the epithelial line demonstrated changes in gene expression consistent with epithelial differentiation. These genes may prove useful as markers for different prostate cell types. The cell lines provide a model system with which to study the process of prostatic epithelial differentiation and stromal-epithelial interactions. This may prove to be useful in the development of differentiation-targeted prostate cancer therapies.     

Breaking through a roadblock in prostate cancer research: an update on human model systems

Prostate cancer is a prevalent disease that affects the aging male population. Whilst there have been significant advances of our biological understanding of the disease, clinical translation of promising agents continues to lag behind. In part, this is due to a paucity of relevant experimental and pre-clinical models required to further develop effective prevention and therapeutic strategies. Genetically modified cell lines fail to entirely represent the genetic and molecular diversity of primary human specimens, particularly from localised disease. Furthermore, primary prostate cancer tissues are extremely difficult to grow in the laboratory and virtually all human models, whether they grow as xenografts in immune-deficient animals or as cell cultures, are genetically modified by the investigator or derived from patients with advanced metastatic disease. In this review, we discuss the latest advances and improvements to current methods of xenografting human primary prostate cancer, and their potential application to translational research.     

Biological properties of human melanoma cells in culture

Summary Three human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and the metastatic melanoma seems to display a higher degree of malignant transformation than the primary. THis work was supported in part by USPHS Grant No. 5 T01 AI00332-06 from the National Institutes of Health, Contract E73-2001-N01-CP-3-3237 from the Virus Cancer Program of the National Cancer Institute, and USPHS Grant No. 0H00714-02 from the National Institute for Occupational Safety and Health.     

Stemness markers characterize IGR-CaP1, a new cell line derived from primary epithelial prostate cancer

Deciphering molecular pathways involved in the early steps of prostate oncogenesis requires both in vitro and in vivo models derived from human primary tumors. However the few recognized models of human prostate epithelial cancer originate from metastases. To date, very few models are proposed from primary tumors and immortalizing normal human prostate cells does not recapitulate the natural history of the disease. By culturing human prostate primary tumor cells onto human epithelial extra-cellular matrix, we successfully selected a new prostate cancer cell line, IGR-CaP1, and clonally-derived subclones. IGR-CaP1 cells, that harbor a tetraploid karyotype, high telomerase activity and mutated TP53, rapidly induced subcutaneous xenografts in nude mice. Furthermore, IGR-CaP1 cell lines, all exhibiting negativity for the androgen receptor and PSA, express the specific prostate markers alpha-methylacyl-CoA racemase and a low level of the prostate-specific membrane antigen PSMA, along with the prostate basal epithelial markers CK5 and CK14. More importantly, these clones express high CD44, CD133, and CXCR4 levels associated with high expression of α2β1-integrin and Oct4 which are reported to be prostate cancer stemness markers. RT-PCR data also revealed high activation of the Sonic Hedgehog signalling pathway in these cells. Additionally, the IGR-CaP1 cells possess a 3D sphere-forming ability and a renewal capacity by maintaining their CSC potential after xenografting in mice. As a result, the hormone-independent IGR-CaP1 cellular clones exhibit the original features of both basal prostate tissue and cancer stemness. Tumorigenic IGR-CaP1 clones constitute invaluable human models for studying prostate cancer progression and drug assessment in vitro as well as in animals specifically for developing new therapeutic approaches targeting prostate cancer stem cells.     

Androgen receptor expression in primary prostate cancers of lobund-wistar rats and in tumor-derived cell lines

Summary Prostate tumors were induced in Lobund-Wistar rats by treatment with N-methyl-N-nitrosourea (MNU) and testosterone propionate (TP). Androgen receptor (AR) expression was confirmed in 16 (100%) of the primary prostate cancers, with strong uniform staining in well-differentiated tumors and more variable AR immunoreactivity in poorly differentiated tumors. Epithelial cell lines were established from nine of the tumors. At early passages, four of the tumor cell lines tested were strongly immunoreactive for AR; however, only two of the cell lines, E2(A) and F2, have remained AR-positive. These cell lines specifically bind ³H-DHT at 40 and 19 fmol/mg protein, respectively, and express a 110 kDa AR immunoreactive protein. Proliferation in in vitro culture of both E2(A) and F2 cells was increased in the presence of 5α-dihydrotestosterone (DHT). The antiandrogen, hydroxyflutamide was able to prevent the DHT-induced growth of E2(A) but not F2 cells. Furthermore, hydroxyflutamide alone increased proliferation of F2 cells, suggesting that the androgen signalling pathway in this cell line may be abnormal. Tumorigenicity of the AR-expressing and nonexpressing cell lines was confirmed by xenograft formation following subcutaneous inoculation into intact male nude mice. In summary, carcinogen-induced prostate tumors of Lobund-Wistar rats express AR and two of nine cell lines derived from the tumors express AR. Further evaluation of AR structure in primary prostate tumors forming spontaneously or following MNU and TP induction will determine whether, as in human prostate cancers, disease progression in Lobund-Wistar rats is associated with mutations in the AR gene.     

Caveolin-1, a metastasis-related gene that promotes cell survival in prostate cancer

Metastasis represents the ultimate target in cancer therapy as this complex biological process is the direct cause of mortality for a variety of human malignancies. The current high level of mortality from prostate cancer results in large part from the inexorable growth of overt or occult metastasis present at the time of diagnosis. Currently, there are no curative therapies for metastatic prostate cancer. To better understand the metastatic phenotype in prostate cancer, we developed a strategy to identify mRNAs that are expressed differentially in cell lines derived from primary versus metastatic mouse prostate cancer using differential display-PCR. In using this system a number of metastasis-related sequences were identified including a cDNA that encodes caveolin-1. Caveolin-1 was found to be overexpressed not only in metastatic mouse prostate cancer, but also in human metastatic disease. Recent studies have indicated that suppression of caveolin-1 expression induces androgen sensitivity in high caveolin-1, androgen-insensitive mouse prostate cancer cells derived from metastases. Conversely, overexpression of caveolin-1 leads to androgen insensitivity in low caveolin, androgen-sensitive mouse prostate cancer cells. Caveolin-1, therefore, is both a metastasis-related gene as well as a candidate androgen resistance gene for prostate cancer in man. Interestingly, recent studies also point to a potential role for caveolin-1 in the resistance of various malignancies to multiple antineoplastic agents. The linkage of caveolin-1 expression with the androgen-resistant phenotype in prostate cancer and the multidrug resistance phenotype in various solid tumors establishes a novel paradigm for understanding these clinically important and now potentially related processes in malignant progression.     

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging. Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.     

Amplification of the Y chromosome in three murine tumor cell lines transformed in vivo by different human prostate cancers

Summary Conventional and molecular cytogenetic analyses of three murine cancer cell lines that had been induced in male athymic mice by the injection of three different human prostate cancer cell lines revealed selective amplification of the Y chromosome. In particular, analysis of metaphase and interphase nuclei by fluorescence in situ hybridization (FISH) with the mouse Y chromosome-specific DNA painting probe revealed the presence of various numbers of Y chromosomes, ranging from one to eight, with a large majority of nuclei showing two copies (46.5–60.1%). In Interphase nuclei, the Y chromosomes showed distinct morphology, allowing identification irrespective of whether the preparations were treated for 15 min or for 5 h with Colcemid, a chemical known to cause chromosome condensation. However, FISH performed on human lymphocyte cultures with chromosome-specific DNA painting probes other than the Y chromosome did not reveal condensed chromosome morphology in interphase nuclei even after 12 h of Colcemid treatment. Our FISH results indicate that (1) the Y chromosome is selectively amplified in all three cell lines; (2) the mouse Y chromosome number is comparable in both interphase and metaphase cells; (3) the Y chromosome number varies between one and eight, with a large majority of cells showing two or three copies in most interphase nuclei; (4) the condensation of the Y chromosome is not affected by the duration of Colcemid treatment but by its inherent DNA constitution; and (5) the number of copies of the Y chromosome is increased and retained not only in human prostate tumor cell lines but also in murine tumors induced by these prostate tumor cell lines.