Potential Tumorigenicity Research Articles

Immortalized Human Fetal Retinal Cells Retain Progenitor Characteristics and Represent a Potential Source for the Treatment of Retinal Degenerative Disease

Human fetal retinal cells have been widely advocated for the development of cellular replacement therapies in patients with retinal dystrophies and age-related macular degeneration. A major limitation, however, is the lack of an abundant and renewable source of cells to meet therapeutic demand, although theoretically this may be addressed through the use of immortalized retinal progenitor cell lines. Here, we have used the temperature-sensitive tsA58 simian virus SV40 T antigen to conditionally immortalize human retinal progenitor cells isolated from retinal tissue at 10-12 weeks of gestation. We show that immortalized human fetal retinal cells retain their progenitor cell properties over many passages, and are comparable with nonimmortalized human fetal retinal cultures from the same gestational period with regard to expression of certain retinal genes. To evaluate the capacity of these cells to integrate into the diseased retina and to screen for potential tumorigenicity, cells were grafted into neonatal hooded Lister rats and RCS dystrophic rats. Both cell lines exhibited scarce integration into the host retina and failed to express markers of mature differentiated retinal cells. Moreover, although immortalized cells showed a greater propensity to survive, the cell lines demonstrated poor long-term survival. All grafts were infiltrated with host macrophage/microglial cells throughout their duration of survival. This study demonstrates that immortalized human fetal retinal progenitor cells retain their progenitor characteristics and may therefore have therapeutic potential in strategies that demand a renewable and consistent supply of donor cells for the treatment of degenerative retinal diseases.

Stem Cell Therapies to Treat Muscular Dystrophy

Muscular dystrophies are heritable, heterogeneous neuromuscular disorders and include Duchenne and Becker muscular dystrophies (DMD and BMD, respectively). DMD patients exhibit progressive muscle weakness and atrophy followed by exhaustion of muscular regenerative capacity, fibrosis, and eventually disruption of the muscle tissue architecture. In-frame mutations in the dystrophin gene lead to expression of a partially functional protein, resulting in the milder BMD. No effective therapies are available at present. Cell-based therapies have been attempted in an effort to promote muscle regeneration, with the hope that the host cells would repopulate the muscle and improve muscle function and pathology. Injection of adult myoblasts has led to the development of new muscle fibers, but several limitations have been identified, such as poor cell survival and limited migratory ability. As an alternative to myoblasts, stem cells were considered preferable for therapeutic applications because of their capacity for self-renewal and differentiation potential. In recent years, encouraging results have been obtained with adult stem cells to treat human diseases such as leukemia, Parkinson's disease, stroke, and muscular dystrophies. Embryonic stem cells (ESCs) can be derived from mammalian embryos in the blastocyst stage, and because they can differentiate into a wide range of specialized cells, they hold potential for use in treating almost all human diseases. Several ongoing studies focus on this possibility, evaluating differentiation of specific cell lines from human ESCs (hESCs) as well as the potential tumorigenicity of hESCs. The most important limitation with using hESCs is that it requires destruction of human blastocysts or embryos. Conversely, adult stem cells have been identified in various tissues, where they serve to maintain, generate, and replace terminally differentiated cells within their specific tissue as the need arises for cell turnover or from tissue injury. Moreover, these cells can participate in regeneration of more than just their specific tissue type. Here we describe multiple types of muscle- and fetal-derived myogenic stem cells, their characterization, and their possible use in treating muscular dystrophies such as DMD and BMD. We also emphasize that the most promising possibility for the management and therapy of DMD and BMD is a combination of different approaches, such as gene and stem cell therapy.

Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors

Reprogramming of differentiated cells into induced pluripotent cells (iPS) was accomplished in 2006 by expressing four, or less, embryonic stem cell (ESC)-specific transcription factors. Due to the possible danger of DNA damage and the potential tumorigenicity associated with such DNA damage, attempts were made to minimize DNA integration by the vectors involved in this process without complete success. Here we present a method of using RNA transfection as a tool for reprogramming human fibroblasts to iPS. We used RNA synthesized in vitro from cDNA of the same reprogramming four transcription factors. After transfection of the RNA, we show intracellular expression and nuclear localization of the respective proteins in at least 70% of the cells. We used five consecutive transfections to support continuous protein expression resulting in the formation of iPS colonies that express alkaline phosphatase and several ESC markers and that can be expanded. This method completely avoids DNA integration and may be developed to replace the use of DNA vectors in the formation of iPS.

Risk of Tumorigenesis in First-in-Human Trials of Embryonic Stem Cell Neural Derivatives: Ethics in the Face of Long-Term Uncertainty

While the successful differentiation of human embryonic stem cells into high purity populations of specialized cells is viewed as a major step towards the safety of prospective human transplantation trials, concerns about the possible risks of tumor formation remain. The potential tumorigenicity of transplants derived from embryonic stem cells raises distinctive ethical challenges regarding trial design, evaluation of their risks to potential benefits ratio, population selection, and post-trial subject monitoring. These challenges are examined within the context of foreseen trials for disorders of the central nervous system.

Genotoxic damage of human adipose-tissue derived mesenchymal stem cells triggers their terminal differentiation

Human adipose tissue-derived mesenchymal (stromal) stem cells (AT-MSCs) and genetically modified to express cytosine deaminase:uracil phosphoribosyltransferase (CDy-AT-MSCs) were treated with hydrogen peroxide in order to induce DNA damage and subsequently evaluate their genetic stability by single cell gel electrophoresis. Both cells types (parental and transgene modified) did not differ in the sensitivity to DNA breaks induction. Potential tumorigenicity of AT-MSCs and CDy-AT-MSCs was tested by subcutaneous inoculation of cell suspension into flank of immunocompromised mice. Dose of 15x10(6) cells was not found to be tumorigenic in given experimental setup. AT-MSCs, CDy-AT-MSCs and MSCs isolated from human lipoma were treated with chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) in attempts to transform them. Surviving cells after genotoxic stress were not transformed but underwent replicative senescence. Irreparable DNA damage caused triggered adipogenic terminal differentiation, rather than apoptosis induction in all kinds of cells tested.

シンポジウム３‐１ 中枢神経系の再生・次なる半世紀 ｉＰＳ細胞技術と神経疾患研究

Induced pluripotent stem (iPS) cells are pluripotent stem cells directly reprogrammed from cultured mouse fibroblast by introducing Oct3/4, Sox2, c-Myc, and Klf4. Cells obtained using this technology, which allows the ethical issues and immunological rejection associated with embryonic stem (ES) cells to be avoided, might be a clinically useful source for cell replacement therapies. We found that secondary neurospheres (SNSs) generated from various mouse iPS cell showed their neural differentiation capacity and teratoma formation after transplantation into the brain of immunodeficient NOD/SCID mice. We found that origin (source of somatic cells) of the iPS cells are the crucial determinant for the potential tumorigenicity of iPS-derived neural stem/progenitor cells and that their tumorigenicity results from the persistent presence of undifferentiated cells within the SNSs. Surprisingly, SNSs derived from c-Myc minus iPS cells generated without drug selection showed robust tumorigenesis, in spite of their potential to contribute adult chimeric mice without tumor formation. Furthermore, we examined whether the transplantation of non-tumorigenic Nanog-iPS-derived SNSs into mouse spinal cord injury (SCI) model could promote locomotor function recovery. As a result, we found that properly pre-evaluated iPS clone-derived neural stem/progenitor cells may be a promising cell source for future transplantation therapy of SCI.

UVA Photoirradiation of Halogenated-Polycyclic Aromatic Hydrocarbons Leading to Induction of Lipid Peroxidation

Since the finding in the 1930s, a large number of polycyclic aromatic hydrocarbons (PAHs) of different structures have been tested for potential tumorigenicity. Structure-activity relationships of halo-PAHs have been investigated to determine the regions of a PAH that may be involved in cancer initiation. From these studies, a number of halo-PAHs were found to be tumorigenic in experimental animals. It was not until the 1980s that halo- PAHs were found to be present in the environment, including municipal incinerator fly ash, urban air, coal combustion, soil, snow, automobile exhausts, and tap water. Due to their widespread presence in the environment and their genotoxic activities, including carcinogenicity, many of these compounds may pose a health risk to humans. Although the biological activities, including metabolism, mutagenicity, and carcinogenicity, of halo- PAHs have been studied their phototoxicity and photo-induced biological activity have not been well examined. In this study, we study the photoirradiation of a series of structure-related halo-PAHs by UVA light in the presence of a lipid, methyl linoleate, and determine as to whether or not these compounds can induce lipid peroxidation. The halo-PAHs chosen for study include 2-bromonaphthalene, 1-chloroanthracene, 9,10- dibromoanthracene, 9-chlorophenanthrene, 9-bromophenanthrene, 7-chlorobenz[a]anthracene, 7- bromobenz[a]anthracene, 7-bromo-5-methylbenz[a]anthracene, 6-chlorobenzo[a]pyrene, and 6- bromobenzo[a]pyrene. The results indicate that upon photoirradiation by UVA all these compounds induced lipid peroxidation at different levels. These results suggest that halo-PAHs may be harmful to human health.

Development of an oral cancer slope factor for Aroclor 1268

Rodent cancer bioassays indicate that substantial differences exist among PCB mixtures in terms of tumorigenic response, although no bioassay has been conducted with Aroclor 1268. The USEPA has used data from these studies to develop three sets of PCB cancer slope factors (CSFs) ranging from 0.07 to 2.0 (mg/kg-day) −1. Selection of the appropriate CSF for risk assessment purposes is largely a function of the exposure circumstances rather than the PCB mixture involved. Since the congener composition of Aroclor 1268 differs substantially from that of the predominant PCB mixture (Aroclor 1254) used to derive the CSFs, the validity of applying existing CSFs to Aroclor 1268 is questionable. We have therefore undertaken the task of developing cancer potency estimates specifically for Aroclor 1268. Potency estimation approaches for Aroclor 1268 were based in part on existing potency estimates for other PCB mixtures, coupled with the relative 2,3,7,8-tetrachlorodibenzo- p-dioxin toxic equivalents (TEQ) content and bioaccumulation potential of PCB mixtures. As such, both Ah-dependent and independent mechanisms of tumorigenesis were considered relevant. Both empirical evidence and mechanistic considerations indicate Aroclor 1268 is substantially less toxic and carcinogenic than the PCB mixtures that have been used by the USEPA to develop CSFs. The present analysis indicates that Aroclor 1268 is likely to be 1–2 orders of magnitude less potent than Aroclor 1254 in terms of potential tumorigenicity. Therefore, we suggest an upper-bound cancer potency factor of 0.27 (mg/kg-day) −1 for Aroclor 1268, a value that is 7- to 8-fold lower than the USEPA’s current default, but nonetheless adequately conservative.

Comparative sequence analysis of the core- and NS5-region of hepatitis C virus from tumor and adjacent non-tumor tissue

The sequence variability within the core- and non-structural 5 (NS5)-region of the hepatitis C virus (HCV) was investigated in tumor and non-tumor tissue of 8 patients with HCV-associated hepatocellular carcinoma (HCC). Analyses of multiple clones containing polymerase chain reaction-amplified HCV sequences revealed a significantly higher variability within the core-region of tumor tissue isolates than of isolates from non-tumor tissue. Mutant sequence diversity ranged from silent mutations, as well as amino acid substitutions, appearance of in frame stop codons and deletions leading to frame-shifts. In contrast, the variability of the NS5-region sequences between isolates from tumor and non-tumor tissue was not significantly different. These observations might have important implications on the pathology of HCV, especially its potential tumorigenicity.

Regulatory issues relating to therapies for periodontal regeneration.

The Food and Drug Administration (FDA) has regulated medical devices since May 1976, when the Medical Device Amendments were enacted. The clinical trial requirements for the marketing of periodontal regeneration devices have been dependent, in part, on the degree of their similarity to devices marketed prior to the legislative enactment date in terms of materials, indication statements, and labeling claims. Nonresorbable barriers were allowed to be marketed based on their equivalence to devices marketed prior to the enactment date based on biocompatability and clinical trial data under the premarket notification requirements section of the law. Bone filling materials such as hydroxyapatite were first marketed based on the finding of equivalence to predicate devices. Newer technologies such as bioabsorbable barriers have also been reviewed under the premarket notification provisions of the law, but manufacturers have been required to provide more extensive safety and effectiveness data to establish equivalence to pre-Amendments devices. Data to answer questions related to the potential toxicity of breakdown products, period of absorption, and ultimate clinical effectiveness needed to be answered prior to marketing. New devices that incorporate technologies that are not substantially equivalent to predicate devices must proceed through the premarket approval route to marketing. For new devices considered a potential significant risk to the patient population, clinical trials are conducted via the investigational device exemption (IDE) requirements that specify the means by which trials will proceed in order to protect the rights of patients. New devices of organic origin, such as bone morphogenic protein, have followed the premarket approval route with IDE requirements instituted as a condition for their path to the marketplace. Issues associated with immediate and long-term contact including potential toxicity, tumorigenicity, and sensitization need to be addressed with appropriate animal models.

Tumorigenic transformation of primary rat embryonal fibroblasts by human papillomavirus type 8 E7 gene in collaboration with the activated H-ras gene.

Particular types of human papillomavirus (HPV) are associated with skin cancer of epidermo‐dysplasia verruciformis (EV) patients. Here, we show, for the first time, that the E7 gene of EV‐associated HPV8 possesses a potential oncogenic transforming ability. The HPV8 E7 open reading frame (ORF) and the HPV16 E7 ORF were cloned under the SV40 promoter/enhancer to construct recombinant plasmids pcD2‐8E7 and pcD2‐16E7, respectively. Transfection of primary rat embryonal flbroblasts having an activated H‐ras gene revealed that pcD2‐8E7 as well as pcD2‐16E7 induced transformation of cells In G418‐resistant colonies at an efficiency of 12.3% and 42.9%, respectively. The resulting transformed cell lines induced by pcD2‐8E7 and activated H‐ras were tumorigenic when injected into syngeneic immunocompetent rats. The potential tumorigenicity of HPV8 E7 seemed to be higher than that of HPV16 E7.

Liquid chromatographic determination of benzo[ a]pyrene at part-per-billion concentrations in highly refined coal- and petroleum-derived fuels

Benzo[ a]pyrene (BaP), a well-known carcinogen, is frequently measured as an “indicator” of the potential dermal tumorigenicity of a sample. The present sequential high-performance liquid chromatography-high-performance liquid chromatography method overcomes problems in trace-level sample enrichment and recovery corrections encountered earlier. An amount of 5 g of naphtha or fuel oil is diluted to 10 ml with dichloromethane and spiked with a small quantity ( ca. 0.25 μg) of 14C-labeled BaP tracer. A BaP-enriched fraction is obtained from a 1-ml aliquot of this sample by semipreparative chromatography on a Partisil PAC 10 column with dichloromethane-hexane (10:90) as the eluent, and concentrated to exactly 0.3, 0.5, or 1.0 ml in acetonitrile. Quantitation is performed using a reversed-phase Vydac 201 TP 5415 column with acetonitrile-water (75:25) as eluent and a Waters Model 420 E/420 AC fluorescence detector, employing an excitation/emission filter pair of 360/425 nm. The recovery of the radiolabeled tracer is evaluated by combustion of 50 μl of the final isolate in pure oxygen, collection of the liberated 14CO 2 in an alkaline desorber, and liquid scintillation counting. The recovery of BaP normally exceeded 90%, but values as low as ca. 50% were occasionally observed. Potential matrix interferences in the recovery determination were eliminated by sample combustion. The nominal precision of the overall method is approximately ± 30% relative standard deviation at a BaP concentration of 30 ppb. The nominal analysis time for a single sample is approximately 4 h.

Toxicological data on NOx: an overview.

This overview is based on experimental and epidemiological studies of NOx toxicity during the past decade. Approximately 130 published studies are cited and about one-fourth of these are discussed briefly under one of the following headings: acute and subacute studies, chronic low-level studies, human studies, and special studies. The latter section examines a selection of comparatively unique investigations, including several devoted to the pulmonary uptake and retention of NO2, and several examining the potential tumorigenicity of NO2. For each major section of the overview, a critical evaluation is attempted in terms of the impact of the appropriate studies on the extant NOx toxicological data base and on the current and planned air quality standards for NOx.

Selective therapy of metastasis. I. Quantitation of tumorigenic circulating and covert cancer cells disseminated from metastatic and nonmetastatic tumors.

To determine the potential efficacy of therapeutic agents and different drug delivery systems against metastases, it is necessary to develop methodologies that can assay the effects of treatment at each step of the metastatic process. This report presents quantitative studies on the total numbers and potential tumorigenicity of cancer cells in the circulation and also secondarily arrested in the lungs of mice following their spontaneous dissemination from methylcholanthrene-induced fibrosarcomas. MC1 fibrosarcomas rarely metastasize but MC2 fibrosarcomas frequently generate pulmonary metastases and it was found, using a combination of direct cancer cell isolation techniques and quantitative bioassays, that MC2 tumors released cancer cells into the bloodstream earlier and with greater frequency than MC1 fibrosarcomas. Also, although similar doses of radiolabeled MC1 and MC2 cells injected intravenously were cleared equally effectively following their arrest in the lung microvasculature, those MC2 cells that survived clearance processes had 10-fold greater lung colonization potential than MC1 cells. Therefore, these experimental systems can be used critically to evaluate the effects of therapeutic agents at individual stages of metastasis as an alternative to measuring their effects solely on solid tumors or the final incidence of metastases, which represents the net result of a number of sequential, complex interactions between cancer cells and the host.