Transformation of human kidney epithelial cells to tumorigenicity by nickel(II) and V-HA-RAS oncogene

Nickel is a toxic metal of environmental concern that has been found to be carcinogenic in man and animals. Primary human kidney (NHKE) cells were immortalized or rescued from senescence after exposure to NiSO₄. The cell lines (IHKE) displayed abnormal karyotype and anchorage independent growth was observed. However, none of the IHKE cells produced tumor upon injection into athymic nude mice. Transfer of the v-Ha-ras oncogene into IHKE cells induced conversion of the immortalized cells into cell lines (THKE) that were tumorigenic when transplanted into athymic nude mice. Ha-ras DNA was present in the transformed cell lines and expressed at high level.     

Molecular heterozygotes in Bacillus subtilis and their correction

Summary Data are presented on the probability of correction of molecular heterozygotes during the transformation of Bacillus subtilis. This value varies between 0 and 1 for different mutants in the same genetic locus. The correction of closely linked markers is simultaneous but it is independent for distant loci. The efficiency of integration of different genetic markers during transformation depends on their ability to be corrected towards the structure of the recipient strain.The linked correction of neighboring markers explains quantitatively the asymmetric phenomena in reciprocal crosses.UV-irradiation of transforming DNA inhibits strongly the correction of molecular heterozygotes and eliminates the asymmetry in reciprocal crosses. The same effect is found after chemical damage of DNA by nitrous acid.     

Spontaneous and nitrosourea-induced primary tumors of the central nervous system in Fischer 344 rats chronically exposed to 836 MHz modulated microwaves.

We have tested an 836.55 MHz field with North American Digital Cellular (NADC) modulation in a 2-year animal bioassay that included fetal exposure. In offspring of pregnant Fischer 344 rats, we tested both spontaneous tumorigenicity and the incidence of induced central nervous system (CNS) tumors after a single dose of the carcinogen ethylnitrosourea (ENU) in utero, followed by intermittent digital-phone field exposure for 24 months. Far-field exposures began on gestational day 19 and continued until weaning at age 21 days. Near-field exposures began at 35 days and continued for the next 22 months, 4 consecutive days weekly, 2 h/day. SAR levels simulated localized peak brain exposures of a cell phone user. Of the 236 original rats, 182 (77%) survived to the termination of the whole experiment and were sacrificed at age 709-712 days. The 54 rats (23%) that died during the study ("preterm rats") formed a separate group for some statistical analyses. There was no evidence of tumorigenic effects in the CNS from exposure to the TDMA field. However, some evidence of tumor-inhibiting effects of TDMA exposure was apparent. Overall, the TDMA field-exposed animals exhibited trends toward a reduced incidence of spontaneous CNS tumors (P < 0. 16, two-tailed) and ENU-induced CNS tumors (P < 0.16, two-tailed). In preterm rats, where primary neural tumors were determined to be the cause of death, fields decreased the incidence of ENU-induced tumors (P < 0.03, two-tailed). We discuss a possible approach to evaluating with greater certainty the possible inhibitory effects of TDMA-field exposure on tumorigenesis in the CNS.     

Sustained early disruption of mitochondrial function contributes to arsenic-induced prostate tumorigenesis

Arsenic is a well-known human carcinogen that affects millions of people worldwide, but the underlying mechanisms of carcinogenesis are unclear. Several epidemiological studies have suggested increased prostate cancer incidence and mortality due to exposure to arsenic. Due to lack of an animal model of arsenic-induced carcinogenesis, we used a prostate epithelial cell culture model to identify a role for mitochondria in arsenic-induced prostate cancer. Mitochondrial morphology and membrane potential was impacted within a few hours of arsenic exposure of non-neoplastic prostate epithelial cells. Chronic arsenic treatment induced mutations in mitochondrial genes and altered mitochondrial functions. Human non-neoplastic prostate epithelial cells continuously cultured for seven months in the presence of 5 µM arsenite showed tumorigenic properties in vitro and induced tumors in SCID mice, which indicated transformation of these cells. Protein and mRNA expression of subunits of mtOXPHOS complex I were decreased in arsenic-transformed cells. Alterations in complex I, a main site for reactive oxygen species (ROS) production as well as increased expression of ROS-producing NOX4 in arsenic-transformed cells suggested a role of oxidative stress in tumorigenic transformation of prostate epithelial cells. Whole genome cGH array analyses of arsenic-transformed prostate cells identified extensive genomic instability. Our study revealed mitochondrial dysfunction induced oxidative stress and decreased expression of p53 in arsenic-transformed cells as an underlying mechanism of the mitochondrial and nuclear genomic instability. These studies suggest that early changes in mitochondrial functions are sustained during prolong arsenic exposure. Overall, our study provides evidence that arsenic disruption of mitochondrial function is an early and key step in tumorigenic transformation of prostate epithelial cells.     

Oxidative DNA damage is a preliminary step during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

The aim of this study was to investigate oxidative DNA damage during 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis. For this purpose, male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12, and 20 weeks. Ten animals were used as negative control. The alkaline Comet assay modified with lesion-specific enzymes was used to detect single and double strand breaks, labile sites (SBs), and oxidised purines and pyrimidines. Although no histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure, oxidative DNA damage was detected in the ‘normal’ oral epithelium. In pre-neoplastic lesions and squamous cell carcinomas induced after 12 and 20 weeks following carcinogen exposure, respectively, oxidative DNA damage was also increased (P < 0.05) when compared to negative control. In conclusion, our results suggest that oxidative DNA damage is an early event during multistep carcinogenesis assay induced by 4NQO. This kind of approach should be considered to persons with high risk of oral cancer, such as in smokers or alcohol consumers.     

Alteration of intracellular GSH levels and its role in microcystin-LR-induced DNA damage in human hepatoma HepG2 cells

Microcystin-LR (MCLR) is a liver-specific toxin known as a tumour promoter in experimental animals. Its mechanisms of hepatotoxicity have been well documented; however, the mechanisms of other effects, in particular those related to its genotoxicity, are not well understood. In our previous studies, we showed that MCLR-induced DNA strand breaks are transiently present and that the damage is mediated by reactive oxygen species (ROS). In this study, we show that exposure of HepG2 cells to non-cytotoxic doses of MCLR-induced time-dependent alterations in the level of intracellular reduced glutathione (GSH). These comprised a rapid initial decrease followed by a gradual increase, reaching a maximum after 6 h of exposure, before returning to the control level after 8 h. During the first 4 h, expression of glutamate-cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis, increased, indicating an increased rate of de novo synthesis of GSH. The most important observation of this study, combined with the results of our previous studies is the correlation between the time course of alterations of intracellular GSH content and the formation and disappearance of MCLR-induced DNA damage. When the intracellular GSH level was reduced, MCLR-induced DNA damage was observed to increase. Later, when the level of intracellular GSH was normal or elevated, new DNA damage was not induced and existing damage was repaired. To confirm the role of GSH system in MCLR-induced genotoxicity, the intracellular GSH level was moderated by pre-treatment with buthionine-(S,R)-sulfoximine (BSO), a specific GSH synthesis inhibitor, and with N-acetylcysteine (NAC), a GSH precursor. Pre-treatment with BSO dramatically increased the susceptibility of HepG2 cells to MCLR-induced DNA damage, while pre-treatment with NAC almost completely prevented MCLR-induced DNA damage. Thus, intracellular GSH is shown to play a critical role in the cellular defence against MCLR-induced DNA damage in HepG2 cells.     

The Weight of Evidence Does Not Support the Listing of Styrene as “Reasonably Anticipated to be a Human Carcinogen” in NTP's Twelfth Report on Carcinogens

Styrene was listed as “reasonably anticipated to be a human carcinogen” in the twelfth edition of the National Toxicology Program's Report on Carcinogens based on what we contend are erroneous findings of limited evidence of carcinogenicity in humans, sufficient evidence of carcinogenicity in experimental animals, and supporting mechanistic data. The epidemiology studies show no consistent increased incidence of, or mortality from, any type of cancer. In animal studies, increased incidence rates of mostly benign tumors have been observed only in certain strains of one species (mice) and at one tissue site (lung). The lack of concordance of tumor incidence and tumor type among animals (even within the same species) and humans indicates that there has been no particular cancer consistently observed among all available studies. The only plausible mechanism for styrene-induced carcinogenesis—a non-genotoxic mode of action that is specific to the mouse lung—is not relevant to humans. As a whole, the evidence does not support the characterization of styrene as “reasonably anticipated to be a human carcinogen,” and styrene should not be listed in the Report on Carcinogens.     

Titanium Dioxide (TiO2) Nanoparticles Preferentially Induce Cell Death in Transformed Cells in a Bak/Bax-Independent Fashion

While the cytotoxic effects of titanium dioxide (TiO₂) nanoparticles have been under intense investigation, the molecular mechanisms of this cytotoxicity remain unknown. Here we investigated the influence of oncogenic transformation and a major apoptotic signaling pathway on cellular responses to TiO₂ nanoparticles. Isogenic wild-type (WT) and apoptosis-resistant (Bak^−/−Bax^−/−) cell lines with and without tumorigenic transformation were examined. TiO₂ nanoparticles preferentially reduced viability of tumorigenic cells in a dose-dependent fashion compared with their untransformed counterparts. Importantly, the elevated cytotoxicity of TiO₂ nanoparticles was independent of a major Bak/Bax-dependent apoptosis pathway. Because transformation does not affect cellular fluid-phase endocytosis or nanoparticle uptake, it is likely that the increased cytotoxicity in tumor cells is due to the interaction between TiO₂ nanoparticles and the lysosomal compartment. Overall, our data indicate that TiO₂ nanoparticles induce cytotoxicity preferentially in transformed cells independent of a major apoptotic signaling pathway.     

Polycomb (PcG) Proteins, BMI1 and SUZ12, Regulate Arsenic-induced Cell Transformation

Inorganic arsenic is a well-documented human carcinogen associated with cancers of the skin, lung, liver, and bladder. However, the underlying mechanisms explaining the tumorigenic role of arsenic are not well understood. The present study explored a potential mechanism of cell transformation induced by arsenic exposure. Exposure to a low dose (0.5 μm) of arsenic trioxide (As(2)O(3)) caused transformation of BALB/c 3T3 cells. In addition, in a xenograft mouse model, tumor growth of the arsenic-induced transformed cells was dramatically increased. In arsenic-induced transformed cells, polycomb group (PcG) proteins, including BMI1 and SUZ12, were activated resulting in enhanced histone H3K27 tri-methylation levels. On the other hand, tumor suppressor p16(INK4a) and p19(ARF) mRNA and protein expression were dramatically suppressed. Introduction of small hairpin (sh) RNA-BMI1 or -SUZ12 into BALB/c 3T3 cells resulted in suppression of arsenic-induced transformation. Histone H3K27 tri-methylation returned to normal in BMI1- or SUZ12-knockdown BALB/c 3T3 cells compared with BMI1- or SUZ12-wildtype cells after arsenic exposure. As a consequence, the expression of p16(INK4a) and p19(ARF) was recovered in arsenic-treated BMI1- or SUZ12-knockdown cells. Thus, arsenic-induced cell transformation was blocked by inhibition of PcG function. Taken together, these results strongly suggest that the polycomb proteins, BMI1 and SUZ12 are required for cell transformation induced by organic arsenic exposure.     

Enhancement of DNA repair using topical T4 endonuclease V does not inhibit melanoma formation in Cdk4R24C/R24C/Tyr-NrasQ61K mice following neonatal UVR

To further investigate the use of DNA repair-enhancing agents for skin cancer prevention, we treated Cdk4^R24C/R24C/Nras^Q61K mice topically with the T4 endonuclease V DNA repair enzyme (known as Dimericine) immediately prior to neonatal ultraviolet radiation (UVR) exposure, which has a powerful effect in exacerbating melanoma development in the mouse model. Dimericine has been shown to reduce the incidence of basal-cell and squamous cell carcinoma. Unexpectedly, we saw no difference in penetrance or age of onset of melanoma after neonatal UVR between Dimericine-treated and control animals, although the drug reduced DNA damage and cellular proliferation in the skin. Interestingly, epidermal melanocytes removed cyclobutane pyrimidine dimers (CPDs) more efficiently than surrounding keratinocytes. Our study indicates that neonatal UVR-initiated melanomas may be driven by mechanisms other than solely that of a large CPD load and/or their inefficient repair. This is further suggestive of different mechanisms by which UVR may enhance the transformation of keratinocytes and melanocytes.     

Endogenous genotoxic agents and processes as a basis of spontaneous carcinogenesis

A list of endogenous DNA-damaging agents and processes is given. Endogenous electrophiles are found with the cosubstrates of physiological transfer reactions (S-adenosylmethionine for methylation, ATP for phosphorylation, NAD+ for ADP-ribosylation, acetyl CoA for acetylation). Aldehyde groups (glyceraldehyde-3-phosphate, formaldehyde, open forms of reducing sugars, degradation products of peroxidation) or alkylating degradation products derived from endogenous nitroso compounds represent additional possibilities. Radical-forming reactions include leakage of the superoxide anion radical from terminal cytochromes and redox cycles, hydroxyl radical formation by the Fenton reaction from endogenous hydrogen peroxide, and the formation of lipid peroxides. Genetic instability by spontaneous deaminations and depurinations as well as replicative instability by tautomer errors and in the presence of mutagenic metal ions represent a third important class of endogenous genotoxic processes. The postulated endogenous genotoxicity could form the mechanistic basis for what is called 'spontaneous' tumor incidence and explain the possibility of an increased tumor incidence after treatment of animals with non-genotoxic compounds exhibiting tumor-promoting activity only. Individual differences are expected to be seen also with endogenous DNA damage. The presence of endogenous DNA damage implies that exogenous DNA-carcinogen adducts give rise to an incremental damage which is expected to be proportional to the carcinogen dose at lowest levels. An increased tumor risk due to exposure to exogenous genotoxic carcinogens could therefore be assessed in terms of the background DNA damage, for instance in multiples of the mean level or of the interindividual variability in a population.     

A diploid rat liver cell culture

Summary Chronic exposure of a cloned rat hepatocyte culture (RL-PR-C) to a subtoxic, sublethal dose of aflatoxin B₁ resulted in malignant transformation. Continuous exposure to aflatoxin B₁ caused increasing tumorigenic potential as tested by back injection into isogenic animals. Control cultures exhibited spontaneous transformation, although approximately 20 passages beyond the chemically induced event. Neither aflatoxin-treated nor control cultures exhibited cytopathological morphology, formation of cell foci, growth in soft agar, or irregular fibroblast-like growth patterns that could be specifically related to the onset of tumorigenic potential. In general, those parameters commonly used to monitor fibroblast cultures for transformation in vitro were not applicable for assessing the tumorigenic potential of these epithelial cells. Karyotypic analyses revealed no specific chromosomal aberrations associated with aflatoxin treatment; however, chromosomal instability was a property of the tumorigenic cell populations. Injection of both aflatoxin-treated and control cultures at passage 56 resulted in tumors indicative of both carcinoma and sarcoma indicating to us the multipotency of these epithelial cells transformed in vitro.     

Chronic low‐dose radiation inhibits cisplatin‐induced formation of tumorous clones in Drosophila melanogaster wts/ + heterozygotes

Ionizing radiation is one of the most extensively studied carcinogens. In contrast to the detrimental effects of high‐dose radiation in carcinogenesis, the biological effects of low‐dose radiation remains poorly understood. In this study, we introduced adult wts/ + heterozygotes of Drosophila melanogaster as transgenic model organisms to determine the tumorigenic activity of low‐dose radiation. The warts (wts) gene is a tumor suppressor gene in mice and humans that is directly involved in cell cycle regulation. Fruit flies at the first larval stage were subjected to ionizing radiation, and then tumorigenic activity was evaluated as the frequency of observed warts tumorous mosaic clones in adult flies. Low‐dose irradiation alone did not cause tumorigenesis in our system. In combined treatment with a chemical carcinogen, chronic irradiation at 0.2 Gy decreased the frequency of tumorous clones induced by cisplatin. These results suggest that low‐dose radiation alone is not deleterious but beneficial in tumorigenesis induced by a chemical carcinogen.     

Karyotypic changes associated with spontaneous acquisition and loss of tumorigenicity in a human transformed bronchial epithelial cell line: Evidence for In vivo selection of transformed clones

Summary In this study, we describe the karyotypic changes associated with the spontaneous acquisition of tumorigenicity in an immortalized tumor bronchial cell line. Neoplastic transformation of the NL20 human bronchial epithelial cell line occurred after 3 yr in culture, and was associated with loss of chromosome 18 together with acquisition of multiple copies of 9q21.2→34. The nontumorigenic NL20 cell line had been established by transfection of human bronchial epithelial cells with the SV40 T antigen, and had retained a relatively stable karyotype after the first 32 passages in vitro. However, when cells from p184 were inoculated into nude mice, a transplantable tumor was obtained that was derived from a minor clone present in this otherwise stable line. Subsequent passaging of the NL20 cells in vitro did not yield further tumors, and the minor clone from which the tumorigenic NL20T cell line derived was no longer evident in NL20 cells by Passage 205. Furthermore, the original tumorigenic NL20T cells lost the neoplastic phenotype after 25 passages in vitro and reverted to the nontumorigenic karyotype observed at p189. In contrast to the loss of the tumorigenic phenotype and karyotype, which occurred with in vitro passaging of the original tumor, when the NL20T cells were passaged in other nude mice, they continued to give rise to tumors with sevenfold amplifications of 9q sequences and loss of chromosome 18, and cells from the secondary tumors (NL20T-A cells) have maintained a stable karyotype and remain tumorigenic even after 64 passages in vitro. A mixture of 10% tumorigenic NL20T-A and 90% nontumorigenic NL20 cells formed tumors in athymic nude mice when cultured in vitro on fibronectin, but not on plastic; cytogenetic analysis demonstrated that the tumors and cell cultures were composed of tumorigenic NL20T-A cells, whereas cytogenetic analysis of cells cultured on plastic were identical to the nontumorigenic NL20 cells. These data support the hypothesis that neoplastic transformation in our original cell line arose from in vivo selection of a small mutant clone, which had arisen in culture and was subsequently selected in vivo but was lost with in vitro culture.     

Tumor-enhancing effects of cholic acid are exerted on the early stages of colon carcinogenesis via induction of aberrant crypt foci with an enhanced growth phenotype.

The objective of the study was to establish whether cholic acid (CHA) enhanced colonic tumor incidence in the early phase of carcinogenesis. Male, Sprague-Dawley rats (n = 180) were injected twice with azoxymethane (AOM) (15 mg x kg(-1) body weight x week(-1), s.c., given 1 week apart). Following the first AOM injection, animals were randomly assigned to two groups, control AIN-93G diet (CON) or control diet containing 0.2% CHA by weight (CHA). Three weeks after the first injection, 20 animals (10 animals/group) were killed and aberrant crypt foci (ACF) were enumerated. The remaining animals were further subdivided and animals randomly assigned to CON or CHA diets, creating four treatments: CON-CON, CON-CHA, CHA-CHA, and CHA-CON. After 3, 12, and 20 weeks (following the first carcinogen injection), the animals were killed and the number and crypt multiplicity of ACF enumerated. Macroscopic tumors were evaluated at week 20. Total ACF were not different between groups. Average crypt multiplicity and medium (4-6 crypts/focus) and large (> or = 7 crypts/focus) ACF were greater in CHA-CHA and CHA-CON compared with CON-CON and CON-CHA (p < 0.01). Transient exposure to CHA (CHA-CON) was sufficient to induce development of ACF with an accelerated growth phenotype and elicit a tumor-enhancing effect. CHA-CHA had the highest tumor incidence (82.8%, p < 0.05) followed by CHA-CON (56.7%, p < 0.05), and tumor multiplicity and number of tumors per rat in CHA-CON were similar to CHA-CHA (2.29 and 1.3 versus 2.33 and 1.9, respectively). Delayed intervention with CHA (CON-CHA) produced a tumor outcome similar to CON-CON (31 and 30%, respectively), it did not enhance colonic tumor incidence. Taken collectively these results suggest CHA was effective in enhancing colon carcinogenesis during early phases and ineffective in post-initiation phases.     

Chemical carcinogen-mouse mammary tumor virus interactions in cell transformation

Summary We have studied the process of mammary cell transformation in vitro using a single cell clone (Clone 18) from a presumptive epithelial cell line, C57MG, derived from a normal mammary gland; a mouse mammary tumor virus (MMTV) host-range variant (RIII)vp4; and the potent initiating carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). After several serial subcultures, cells treated with virus and then with carcinogen exhibited an altered (transformed) morphology, a dramatic increase in anchorage independence, an increase in multinucleation after exposure to cytochalasin B, an enhanced ability to proliferate in low Ca²⁺ (0.01 mM) medium, and tumorigenicity when inoculated subcutaneously into athymic (nude) mice. Although some of these phenotypic alterations were observed also in cultures treated singly with MMTV or DMBA and in cultures exposed to DMBA before infection with MMTV, enhanced cytochalasin B multinucleation and tumorigenicity were properties observed only in mass cultures of cloned cells first infected with MMTV and then exposed to DMBA. This demonstrates for the first time that exposure of presumptive mammary epithelial cells to MMTV followed by DMBA, but not to either agent alone or to DMBA followed by MMTV, results in malignant transformation of these cells. Support for these studies was provided in part by the National Cancer Institute, Bethesda, Md., Contract N01-CP-01018.     

Heavy ion mutagenesis: Linear energy transfer effects and genetic linkage

We have characterized a series of 69 independent mutants at the endogenoushprt locus of human TK6 lymphoblasts and over 200 independent S 1-deficient mutants of the humanxhamster hybrid cell line A_L arising spontaneously or following low-fluence exposures to densely ionizing Fe ions (600 MeV/amu, linear energy transfer = 190 keV/µm). We find that large deletions are common. The entirehprt gene (>44 kb) was missing in 19/39 Fe-induced mutants, while only 2/30 spontaneous mutants lost the entirehprt coding sequence. When the gene of interest (S1 locus = M1C1 gene) is located on a nonessential human chromosome 11, multilocus deletions of several million base pairs are observed frequently. The S1 mutation frequency is more than 50-fold greater than the frequency ofhprt mutants in the same cells. Taken together, these results suggest that low-fluence exposures to Fe ions are often cytotoxic due to their ability to create multilocus deletions that may often include the loss of essential genes. In addition, the tumorigenic potential of these HZE heavy ions may be due to the high potential for loss of tumor suppressor genes. The relative insensitivity of thehprt locus to mutation is likely due to tight linkage to a gene that is required for viability.Invited paper presented at the International Symposium on Heavy Ion Research: Space, Radiation Protection and Therapy, Sophia-Antipolis, France, 21–24 March 1994     

DNA repair and the evolution of transformation IV. DNA damage increases transformation

Natural genetic transformation in the bacterium Bacillus subtilis provides a model system to explore the evolutionary function of sexual recombination. In the present work, we study the response of transformation to UV irradiation using donor DNAs that differ in sequence homology to the recipient's chromosome and in the mechanism of transformation. The four donor DNAs used include homologous-chromosomal-DNA, two plasmids containing a fragment of B. subtilis trp⁺ operon DNA and a plasmid with no sequence homology to the recipient cell's DNA. Transformation frequencies for these DNA molecules increase with increasing levels of DNA damage (UV radiation) to recipient cells, only if their transformation requires homologous recombination (i.e. is recA⁺-dependent). Transformation with non-homologous DNA is independent of the recipient's recombination system and transformation frequencies for it do not respond to increases in UV radiation. The transformation frequency for a selectable marker increases in response to DNA damage more dramatically when the locus is present on small, plasmid-borne, homologous fragments than if it is carried on high molecular weight chromosomal fragments. We also study the kinetics of transformation for the different donor DNAs. Different kinetics are observed for homologous transformation depending on whether the homologous locus is carried on a plasmid or on chromosomal fragments. Chromosomal DNA- and non-homologous-plasmid-DNA-mediated transformation is complete (maximal) within several minutes, while transformation with a plasmid containing homologous DNA is still occurring after an hour. The results indicate that DNA damage directly increases rates of homologous recombination and transformation in B. subtilis. The relevance of these results and recent results of other labs to the evolution of transformation are discussed.     

Enumeration of the simian virus 40 early region elements necessary for human cell transformation

While it is clear that cancer arises from the accumulation of genetic mutations that endow the malignant cell with the properties of uncontrolled growth and proliferation, the precise combinations of mutations that program human tumor cell growth remain unknown. The study of the transforming proteins derived from DNA tumor viruses in experimental models of transformation has provided fundamental insights into the process of cell transformation. We recently reported that coexpression of the simian virus 40 (SV40) early region (ER), the gene encoding the telomerase catalytic subunit (hTERT), and an oncogenic allele of the H-ras gene in normal human fibroblast, kidney epithelial, and mammary epithelial cells converted these cells to a tumorigenic state. Here we show that the SV40 ER contributes to tumorigenic transformation in the presence of hTERT and oncogenic H-ras by perturbing three intracellular pathways through the actions of the SV40 large T antigen (LT) and the SV40 small t antigen (ST). LT simultaneously disables the retinoblastoma (pRB) and p53 tumor suppressor pathways; however, complete transformation of human cells requires the additional perturbation of protein phosphatase 2A by ST. Expression of ST in this setting stimulates cell proliferation, permits anchorage-independent growth, and confers increased resistance to nutrient deprivation. Taken together, these observations define the elements of the SV40 ER required for the transformation of human cells and begin to delineate a set of intracellular pathways whose disruption, in aggregate, appears to be necessary to generate tumorigenic human cells.     

Overview of the spaceflight radiation environment and its impact on cell biology experiments

Variables studied in typical cellular radiation biology experiments are cell killing, mutagenesis, transformation to malignancy, heritable damage, and DNA damage and repair. Dose response curves for cells exposed to low-LET radiations and some high LET radiations are well known. The low-LET dose rate in low earth orbit is roughly 1.0 mSv/day, the heavy-ion (Z>2) flux is about 1.0 particle/cm2-s corresponding to about 0.3 mSv/day, and the integrated neutron flux is roughly 2 neutrons/cm2-s corresponding to 0.012 mGy/d or, assuming a QF of 10, 0.12 mSv/d. Published dose-response curves were used to estimate the probability that a mammalian cell will be affected by each of the above types of damage. As a general approximation the exposure of an experimental cell population to the space radiation environment for 100 days will result in the following probabilities of damage per cell: cell killing based on clonogenicity 0.02, mutagenesis per locus based on phenotype analysis 1 x 10(-6), point mutation induction 2 x 10(-8) per locus, malignant transformation in vitro based on colony morphology 1.2 x 10(-5), heritable damage based on colony size 0.02, and induced DNA double-strand breaks based on fragment analysis by electrophoresis 3.5/cell or 0.26/cell after repair. Most of these figures are accurate to within a factor of 2. Thus the spaceflight radiation environment has essentially undetectable impact on typical cell biology experiments unless experimental goals involve the precise measurement of one of the above end-points. Other in vitro end-points, such as tissue morphogenesis and cell differentiation, are expected to be similarly unaffected by the spaceflight radiation environment.