Guidelines for the use of cell lines in biomedical research.

Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

A novel murine model of allogeneic vaccination against prostate cancer.

Prostate cancer continues to be a major cause of death in men. Surgical and medical treatments of the disease have improved, but metastasic disease remains a significant clinical problem. Novel therapies such as whole cell vaccination offer the potential of treating disease by stimulating the immune system. To study the efficacy of a whole cell vaccine in prostate cancer two strains of mice were used: C57BL/6 (H-2Kb) and C3H/HeJ (H-2K(k)) in combination with four different cell lines. Thus, a model was constructed of allogeneic and syngeneic vaccine, as well as a challenge tumour for each strain. Two novel cell lines were developed during this study. Firstly, the non tumourigeneic PMC-1 was derived from a normal mouse prostate and immortalized with HPV16. Secondly, the tumourigeneic PMC-1 C6ras1p1 was transformed with human ras gene which formed tumours in both SCID and C3H/HeJ mice. Protection, and the nature of the immune response to syngeneic and allogeneic vaccine, in males and females was examined in both strains. Vaccination with both syngeneic and allogeneic irradiated whole cell vaccines induced protection from syngeneic challenge in females. However, no protection was observed when allogeneic vaccine was given to male mice. This correlated with the immune response. Two types of cellular immune responses were generated in females. A NK-mediated response was observed in C57BL/6 mice, whilst C3H/HeJ mice developed a CTL response. Little or no cellular immune response was observed in males. The cytokine profile in C3H/HeJ females was a mixture of Th1 and Th2 whilst a mainly Th1 profile was observed in C57BL/6 mice. Male mice showed a diminished cytokine secretion compared to females which was further depressed after challenge. The difference in immunity was largely as expected, since tolerance to prostate antigens should not normally develop in female mice. However, this makes this model particularly relevant clinically since it directly mimics the human situation and thus may accelerate the development of whole cell vaccines for clinical use.

A human prostatic stromal myofibroblast cell line WPMY-1: a model for stromal-epithelial interactions in prostatic neoplasia.

Here we report the characterization of an SV40 large-T antigen-immortalized stromal cell line, WPMY-1, derived from the same prostate as our previously described epithelial cell lines. The WPMY-1 cells were determined to be myofibroblasts on the basis of co-expression of smooth muscle alpha-actin and vimentin. They also show positive staining for androgen receptor, large-T antigen, and positive but heterogeneous staining for p53 and pRb. Their growth is stimulated by the synthetic androgen mibolerone to 145% of control (100%). Platelet-derived growth factor BB, epidermal growth factor and basic fibroblast growth factor, at 10 ng/ml, stimulated growth to 138, 143 and 146% of control, respectively. Transforming growth factor-beta, at 10 ng/ml, inhibited serum-induced growth to 65% of control in the presence of 1% serum, and bFGF-induced growth to 30% of control. A serum-free medium was developed for optimal growth of WPMY-1 cells. They show anchorage-independent growth in soft agar. Studies on paracrine interactions show that myofibroblast-conditioned medium causes a marked inhibition of growth in WPE1-10 cells, while conditioned medium from WPE1-10 prostatic epithelial cells caused only a small increase in the growth of WPMY-1 cells. WPMY-1 cells secrete very low levels of MMP-9 but high levels of MMP-2, markedly higher than the epithelial cells. These epithelial and myofibroblast cell lines, derived from the same prostate, provide novel and useful models for studies on paracrine stromal-epithelial interactions in carcinogenesis, tumor progression, prevention and treatment of prostate cancer and benign prostatic hyperplasia.

Protein changes associated with ionizing radiation-induced apoptosis in human prostate epithelial tumor cells.

Ionizing radiation (IR) is an important component in the therapy of localized prostate cancer. Identification of protein alterations during IR-induced apoptosis prostate cancer cells is an important step toward understanding the new metabolic status of the dying cell. In the present study, we report changes in protein profile that define the execution phase of the apoptotic response in the in vitro model of tumorigenic radiation-transformed SV40-immortalized human prostate epithelial cells (267B1-XR), induced to undergo programmed cell death by IR. We employed an approach that involves use of analytical two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) coupled with Western blotting with specific antisera. Our results point out that apoptotic cells experience significant reduction in the levels of the intermediate filament proteins, keratins-18, 19, vimentin and the associated 14-3-3 adapter proteins. At the same time, molecular chaperones such as glucose-regulated protein 94, calreticulin, calnexin, and protein disulfide isomerase exhibit marked accumulation in these dying cells. The present data indicate that apoptosis-associated processes in prostate epithelial cells include solubilization of the rigid intermediate filament network by specific proteolysis as well as increased levels of endoplasmic reticulum (ER) proteins with chaperone functions.

Differential expression of stathmin during neoplastic conversion of human prostate epithelial cells is reversed by hypomethylating agent, 5-azacytidine.

In a variety of human tumor tissues, including those of prostate and breast, CpG hypermethylation represents one of the mechanisms downregulating the expression of specific proteins, including tumor suppressor proteins. Using 267B1-XR cells generated by ionizing radiation-induced transformation of epithelial cells, derived from neonatal human prostate and immortalized by SV40 (267B1), we now report markedly low levels of expression of the cytoplasmic phosphoprotein stathmin, in addition to several proteins of the actin microfilaments and intermediate filaments that characterize the altered phenotype. Stathmin is emerging as a relay protein integrating signals from diverse pathways during differentiation and neoplastic progression. In this in vitro prostate carcinogenesis model system, where loss of specific-protein expression is a major feature of the transformed 267B1-XR cells, we employed 5-azacytidine treatment followed by 2D-PAGE to reveal if experimental genomic hypomethylation reinstated the levels of any of the differentially expressed proteins. Our data suggest that stathmin represents one such example.

Apoptosis-associated proteolysis of vimentin in human prostate epithelial tumor cells.

Vimentin intermediate filaments (IF) are responsible for regulation of cell attachment and subcellular organization. Using an in vitro model system of human prostate epithelial cells (267B1-XR), we demonstrate that a series of vimentin proteolytic fragments represent some of the differentially expressed proteins in 2D-gel profiles of the apoptotic cells undergoing ionizing radiation-induced cell death. A caspase-sensitive motif search suggests that the type III IF protein (vimentin) is subject to proteolysis to promote the execution phase of apoptosis, in a manner similar to the well-established type V (lamins) and type I (keratins 18, 19) IF proteins. Furthermore, vimentin and a few of its derived polypeptides, reported to be specific to the apoptotic process, correspond to ubiquinated proteins, thus pointing to the complex interrelationships of protein ubiquination in solubilizing the IF network during apoptosis.

Expression of cytokeratin-19 as a marker of neoplastic progression of human prostate epithelial cells.

BACKGROUND: Our earlier studies demonstrated neoplastic transformation of SV40-immortalized neonatal human prostate epithelial cells (267B1) by fractionated doses of ionizing radiation or by introduction of v-ki-ras oncogene. X-ray-treated 267B1 cells represent three different stages of neoplastic progression: nontumorigenic F3-SAC cells that acquired morphological changes and anchorage independence when treated with 2 x 2 Gy of X-rays; malignantly transformed 267B1-XR and 267B1-SXR cells that received 2-Gy doses to a total of 30 Gy. We also reported alterations in cell size, morphology, actin stress fibers, and levels of actin-binding proteins in these transformed human prostate cells. METHODS: We analyzed intermediate filament-nuclear matrix (IF-NM) protein expression in the various 267B1 cells as a consequence of neoplastic progression by two-dimensional gel electrophoresis and immunofluorescence. RESULTS: Our present study revealed that the 267B1 cells experienced progressive changes in their intermediate filament protein composition during the process of neoplastic conversion, achieved either by X-rays or by ras-oncogene. In particular, we observed a stepwise downregulation of cytokeratin-19 in these in vitro transformed 267B1 cells. CONCLUSIONS: Our data suggest that loss of expression of cytokeratin-19 accompanied the morphological alterations associated with in vitro neoplastic transformation of SV40-immortalized prostate epithelial cells.

Protein expression changes associated with radiation-induced neoplastic progression of human prostate epithelial cells.

Carcinogenic progression in most epithelial systems is a multistep process and presents as numerous (un)stable intermediate stages prior to the development of a fully malignant phenotype. Recently, we reported the neoplastic transformation of an SV40 immortalized, neonatal human prostate epithelial cell line (267B1) by multiple exposures to X-rays [1, 2]. The parental 267B1 cells acquired anchorage-independence and exhibited morphological transformation following exposure to two consecutive doses of 2 Gy. Exposure of either the parental 267B1 cells or the anchorage-independent derivatives (F3-SAC) to a total dose of 30 Gy of X-rays yielded tumorigenic transformants (267B1-XR and 267B1-SXR, respectively). All of these radiation-treated derivatives (F3-SAC, 267B1-XR, and 267B1-SXR) were characterized by reduced cell size and poorly organized actin stress fibers [2, 3]. The present study examines the protein expression changes associated with cytoskeletal alterations during the different steps of neoplastic progression induced by X-rays in the in vitro human prostate cell system. This analysis was achieved by using the high resolving power of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) in the 267B1, F3-SAC, 267B1-XR, and 267B1-SXR cells. We report changes in the expression of gelsolin in the partially transformed, anchorage-independent, nontumorigenic (F3-SAC) cells and a progressive loss of expression of tropomyosin isoforms (TM-1 and TM-3), and myosin light chain-2 (MLC-2) in the tumorigenic (267B1-XR; 267B1-SXR) cells, respectively. In contrast, our results demonstrate that the levels of the small GTP-binding protein Rho-A, an active participant in the actin stress fiber organization, are not altered during neoplastic progression of these 267B1 cells. Thus the changes in synthesis of gelsolin, tropomyosins, and MLC-2 provide a rationale for the alterations in the actin stress fiber formation and reduction in cell size during the exposure of prostate epithelial cells to multiple doses of X-rays.

Malignant transformation of human prostate epithelial cells by N-nitroso-N-methylurea.

We report the malignant transformation of adult human prostate epithelial cells after multiple exposures to the chemical carcinogen N-nitroso-N-methylurea. Such transformants showed morphological alterations and anchorage-independent growth in soft agar and induced carcinomas when transplanted into nude mice. No p53 or ras mutations were observed. Stepwise chromosomal changes in the progression to tumorigenicity were observed. Loss of the p arms of chromosome 8 (p10>pter) and chromosome 10 (p10>pter) and gain of the q arm of chromosome 8 (q10>qter) were only observed in the tumor outgrows. These findings provide the first evidence of malignant transformation of human prostate epithelial cells exposed to a chemical carcinogen.

Chromosomal changes observed in immortalized human keratinocytes transformed by ionizing radiation.

Human epithelial cancer cells were induced by concerted action of DNA tumor virus and X-ray radiation. Treatment of nontumorigenic early passage AD12-SV40 immortalized epithelial cells (RHEK-1) at passage 23 with radiation, resulted in further changes in their growth properties. One day old cultures of these RHEK-1 cells were irradiated with graded doses of X-rays (0, 2, 4, 6, and 8 Gy i.e. RHEK-1, RHEK-1/200R, RHEK-1/400R, RHEK-1/600R, and RHEK-1/800R). Morphologic alterations, the ability to grow in soft agar, and to form rapidly-growing squamous cell carcinomas in nude mice were concomitantly acquired properties of the radiation transformed cell lines RHEK-1/200R and RHEK-1/ 400R. On the basis of commonality in having addition of some extra material in chromosome 11 in the region between q14/q22 in all tumorigenic cell lines RHEK-1/200R and RHEK-1/400R, and deletion of the same region in nontumorigenic irradiated cell lines-RHEK-1/600R and RHEK-1/800R, it is deduced this region may have some important oncogene/s or other gene/s that play an important role in tumorigenesis. When compared to squamous cell carcinoma data, the duplication observed in the present study is also observed in 28 to 38% of head and neck and also in 25% of cases of untreated malignant lesions of oral squamous cell carcinoma. Thus, this study shows the correlation between in vitro induced squamous cell carcinoma to in vivo tumors.

Oncogenic activation of the tyrosine kinase domain of the human trk proto-oncogene by fusion to a cell adhesion molecule.

To investigate the mechanisms of radiation-induced neoplastic conversion, DNA from X-ray transformed human epidermal keratinocytes (RHEK-1) was used in sequential cycles of NIH3T3 transfection followed by nude mice tumorigenicity assays. NIH3T3-derived transformants retained discrete DNA fragments hybridizing to human alu probes. Four clones were isolated from a cosmid library prepared from one of these transformants (49-7G) using human DNA as the probe. Analyses of DNAs from 49-7G cells and the four cosmid clones with probes for a number of human oncogenes demonstrated that the cloned sequences were related to the trk oncogene. Transfection of NIH3T3 cells with the cosmid DNAs did not result in the appearance of transformed foci when the murine fibroblasts were cultured on plastic. However, foci developed when transfected cells were cultured on plates coated with various extracellular matrix (ECM) components. Neomycin-resistant cosmid-transfected NIH3T3 cells did induce tumors in nude mice, and their tumorigenicity correlated with their level of trk expression. Nucleotide sequence analyses of cDNA clones isolated from a 49-7G library with a human trk probe revealed that the cloned sequences resulted from the fusion between 5' sequences from the human beta-1,4-galactosyltransferase gene, which encodes a membrane protein involved in cell-cell and cell-matrix interactions, and 3' sequences from the human trk proto-oncogene. The 76 kDa protein product of the chimeric gene, designated bgt-trk, has been identified in NIH3T3 cells transfected with cosmid 19/2 or with bgt-trk cDNA expression constructs, and its phosphorylation in tyrosine has been found to increase when the transfected cells were seeded on plates coated with ECM components which also elicited foci formation in NIH3T3 transformation assays. The fusion of the trk tyrosine kinase domain to a cell adhesion molecule may explain the ECM dependence for the expression of the full transforming potential of the resulting oncogene product.

Radiation-induced neoplastic transformation of human prostate epithelial cells.

We report the malignant transformation of human prostate epithelial cells (267B1) after multiple exposures to ionizing radiation. Carcinogenic progression of cells from immortal growth to anchorage-independent growth in soft agar to tumorigenicity in athymic mice resulted after a cumulative X-ray dose of 30 Gy. The tumors were characterized histologically as poorly differentiated adenocarcinomas, expressed prostate-specific antigen, and stained positive for keratin. No p53 or ras mutations were observed. Numerous chromosomal defects were noted on karyotypes after radiation exposure. However, chromosome 3 and 8 translocations were observed predominantly in the tumor outgrowths. These findings provide the first evidence of malignant transformation of human prostate epithelial cells exposed to ionizing radiation.

Cytoskeletal changes during radiation-induced neoplastic transformation of human prostate epithelial cells.

We recently reported tumorigenic transformation of SV40-immortalized neonatal human prostate epithelial cells (267B1) by exposure to fractionated doses of X-rays. Altered morphology and anchorage independence were observed following two successive fractions of 2 Gy each (F3-SAC). Additional 2 Gy treatments to these non-tumorigenic cells to a total dose of 30 Gy resulted in radiation-transformed tumorigenic colonies (267B1-SXR). Malignant transformation of parental 267B1 cells was also achieved by consecutive 2 Gy exposures to a total dose of 30 Gy (267B1-XR). This study discusses the cytoskeletal changes in the F3-SAC, 267B1-XR and 267B1-SXR derivatives of these human prostate epithelial cells. Confocal and conventional fluorescence microscopy of filamentous actin showed numerous, well organized, evenly distributed stress fibers in the parental cells prior to irradiation, while the anchorage-independent cells and several tumorigenic derivatives exhibited poor stress fiber organization after radiation exposure. This disorganization of actin microfilaments in the radiation-transformed cells was also accompanied by changes in the expression of selective tropomyosin isoforms as judged by two-dimensional gel electrophoresis. These changes in actin organization and tropomyosin expression appear to be coincidental with morphological transformation and acquisition of tumorigenicity in the 267B1 cells following radiation exposure.

Co-regulated expression of dbl and poly(ADP-ribose) polymerase in Ewing's sarcoma cells and dbl-transformed NIH3T3 fibroblasts.

Poly(ADP-ribose) polymerase (PADPRP) is a ubiquitous enzyme constitutively expressed at low levels in the majority of eukaryotic cells, including most mammalian tumors and tumor-derived cell lines. Overexpression of PADPRP following the introduction of cDNA recombinant constructs into various cell types results in cell death. An exception to this effect are Ewing's sarcoma (ES) cells, which have been shown to contain elevated steady-state levels of PADPRP mRNA and high constitutive levels of protein and polymerase activity. In fact, this excess of PADPRP has been suggested to participate in the intrinsic radiosensitivity of Ewing's sarcomas, a highly malignant childhood bone tumor frequently curable with radiotherapy. It appears that ES cells might possess a hitherto unknown mechanism(s) by which PADPRP overexpression is controlled and made compatible with cell survival and proliferation. In order to investigate the contribution of other genetic alterations to PADPRP regulation in ES cells, we analysed the expression levels of PADPRP and of other genes, such as oncogenes and tumor suppressor genes, which may enhance the proliferative potential of ES cells. We have detected a positive correlation between the expression levels of the DNA-repair enzyme poly(ADP-ribose) polymerase and the dbl proto-oncogene in Ewing's sarcoma cells. The co-regulated expression of these genes has been established in NIH3T3 cells transformed by the human dbl oncogene or by overexpression of the dbl proto-oncogene. In both instances, the increase in dbl expression resulted in elevated levels of PADPRP mRNA and polymerase activity. The dbl oncogene was more efficient than the proto-oncogene in upregulating PADPRP expression. The inability of other oncogenes to upregulate PADPRP upon transformation of NIH3T3 cells demonstrated the specificity of the dbl in the process. Transfection of dbl-transformed NIH3T3 cells with retroviral PADPRP vectors resulted in the establishment of clones with PADPRP levels higher than those detectable in untransformed NIH3T3 cells transfected with the same retroviral constructs. These results suggest that dbl plays a role in the mechanism by which mammalian cells autoregulate their endogenous levels of PADPRP. Post-translational modification of the dbl or proto-dbl proteins by cytoplasmic PADPRP does not participate in the mechanism(s) underlying the observed PADPRP/dbl co-regulation.

Transformation of human epidermal keratinocytes with fission neutrons.

The biological effects of exposures to high LET radiations have particular relevance to radiation protection and risk assessment. Since most cancers are of epithelial origin, it is important to obtain a better understanding of radiation-induced oncogenic transformation in this cell type. Accordingly we have initiated studies to determine whether immortalized human epidermal keratinocytes (RHEK) can be transformed with high LET radiations. Exponentially growing RHEK cells were treated with single doses (1, 10, 25, 50 and 100 cGy) of 0.85 MeV fission neutrons from the Janus reactor. Neutron exposure led to the development of morphologically altered cells and foci formation after 6 weeks at confluence. These transformed cultures grew with an increased saturation density, exhibited anchorage-independent growth and formed tumors in athymic mice. Single-strand conformational polymorphism analysis and DNA sequencing demonstrated the absence of point mutations in codons 12/13 and 61 in the Ha-ras, Ki-ras, or N-ras genes and exons 4-9 of the p53 tumor suppressor gene. These studies demonstrate that high LET radiations (fission neutrons) can transform immortalized human epithelial cells to a malignant phenotype that does not appear to involve mutations in either the cellular p53 or ras genes.

Radiation-induced neoplastic transformation of human cells.

Ionizing radiation can induce cancers in humans and animals and can cause in vitro neoplastic transformation of various rodent cell systems. However, numerous attempts to achieve neoplastic transformation of human cells by radiation have generally proven unsuccessful. Neoplastic transformation of immortalized human epidermal keratinocytes by X-ray irradiation has recently been reported. The carcinogenic effect of radiation on cultured human cells will be briefly reviewed. The current state-of-the-art in radiation-induced transformation of human cells in culture is presented. This will provide insight into the molecular and cellular mechanisms in the conversion of normal cells to a neoplastic state of growth.

Neoplastic transformation of a human prostate epithelial cell line by the v-Ki-ras oncogene.

Investigations of mechanisms of human prostate carcinogenesis are limited by the unavailability of a suitable in vitro model system. We have demonstrated that an immortal, but nontumorigenic, human epithelial cell line (267B1) established from fetal prostate tissue can be malignantly transformed by a biological carcinogen, and can serve as a useful model for investigations of the progression steps of carcinogenesis. Activated Ki-ras was introduced into 267B1 cells by infection with the Kirsten murine sarcoma virus. Morphological alterations and anchorage-independent growth were observed; when cells were injected into nude mice, poorly differentiated adenocarcinomas developed. These findings represent the first evidence of malignant transformation of human prostate epithelial cells in culture, and support a role for Ki-ras activation in a multistep process for prostate neoplastic transformation.

Neoplastic transformation of immortalized human keratinocytes by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most powerful carcinogen ever tested in animals. Recent epidemiological studies have suggested its carcinogenic potential in humans. In the present study, nontumorigenic human epidermal keratinocytes immortalized by adenovirus 12-simian virus 40 (Ad12-SV40) were transformed by exposures of TCDD equal to or greater than 0.1 nM for 2 wk. These transformed cells showed morphological alterations and induced carcinomas when transplanted into nude mice, whereas no such transformation phenotypes were observed with exposures of less than 0.1 nM for 2 wk. Primary human epithelial keratinocytes exposed to various concentrations of TCDD failed to show any evidence of transformation. Induction of aryl hydrocarbon hydroxylase activity was dose dependent, as was transformation. Thus, the carcinogenicity of TCDD in this human cell system appears to be an Ah receptor-mediated process. The present study represents the first evidence of neoplastic conversion of human cells exposed to this environmentally important chemical.

Baseline sister chromatid exchange in human cell lines with different levels of poly(ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase is a chromatin enzyme which adds long chains of ADP-ribose to various acceptor proteins in response to DNA strand breaks. Its primary function is unknown; however, a role in DNA repair and radiation resistance has been postulated based largely on experiments with enzyme inhibitors. Recent reports of mutant cell lines, deficient in poly(ADP-ribose) polymerase activity, have supported previous studies with inhibitors, which suggests the involvement of poly(ADP-ribose) polymerase in maintaining baseline levels of sister chromatid exchanges. Mutant cells with even slightly depressed enzyme levels show large elevation of baseline sister chromatid exchanges. Since intracellular poly(ADP-ribose) polymerase levels can vary greatly between different nonmutant cell lines, we surveyed levels of baseline sister chromatid exchange in normal and tumor human cell lines and compared them with endogenous levels of poly(ADP-ribose) polymerase. Despite 10-fold differences in poly(ADP-ribose) polymerase, the baseline level of sister chromatid exchanges remained relatively constant in the different cell lines (0.13 +/- 0.03 SCE/chromosome), with no indication of a protective effect for cells with high levels of the enzyme.