Epperly, M. W., Gretton, J. A., DeFilippi, S. J., Sikora, C. A., Liggitt, D., Koe, G. and Greenberger, J. S. Modulation of Radiation-Induced Cytokine Elevation Associated with Esophagitis and Esophageal Stricture by Manganese Superoxide Dismutase-Plasmid/Liposome (SOD2-PL) Gene Therapy.

Radiation of the esophagus of C3H/HeNsd mice with 35 or 37 Gy of 6 MV X rays induces significantly increased RNA transcription for interleukin 1 (Il1), tumor necrosis factor alpha (Tnf), interferon gamma inducing factor (Ifngr), and interferon gamma (Ifng). These elevations are associated with DNA damage that is detectable by a comet assay of explanted esophageal cells, apoptosis of the esophageal basal lining layer cells in situ, and micro-ulceration leading to dehydration and death. The histopathology and time sequence of events are comparable to the esophagitis in humans that is associated with chemoradiotherapy of non-small cell lung carcinoma (NSCLC). Intraesophageal injection of clinical-grade manganese superoxide dismutase-plasmid/liposome (SOD2-PL) 24 h prior to irradiation produced an increase in SOD2 biochemical activity in explanted esophagus. An equivalent therapeutic plasmid weight of 10 μg ALP plasmid in the same 500 μl of liposomes, correlated to around 52–60% of alkaline phosphatase-positive cells in the squamous layer of the esophagus at 24 h. Administration of SOD2-PL prior to irradiation mediated a significant decrease in induction of cytokine mRNA by radiation and decreased apoptosis of squamous lining cells, micro-ulceration, and esophagitis. Groups of mice receiving 35 or 37 Gy esophageal irradiation by a technique protecting the lungs and treating only the central mediastinal area were followed to assess the long-term effects of radiation. SOD2-PL-treated irradiated mice demonstrated a significant decrease in esophageal wall thickness at day 100 compared to irradiated controls. Mice with orthotopic thoracic tumors composed of 32D-v-abl cells that received intraesophageal SOD2-PL treatment showed transgenic mRNA in the esophagus at 24 h, but no detectable human SOD2 transgene mRNA in explanted tumors by nested RT-PCR. These data provide support for translation of this strategy of SOD2-PL gene therapy to studies leading to a clinical trial in fractionated irradiation to decrease the acute and chronic side effects of radiation-induced damage to the esophagus.

Pogue, B. W., O'Hara, J. A., Wilmot, C. M., Paulsen, K. D. and Swartz, H. M. Estimation of Oxygen Distribution in RIF-1 Tumors by Diffusion Model-Based Interpretation of Pimonidazole Hypoxia and Eppendorf Measurements.

Numerical simulations of oxygen diffusion from the capillaries in tumor tissue were used to predict the capillary oxygen supply within and near hypoxic regions of the RIF-1 tumor. A finite element method to simulate the oxygen distribution from a histology section is presented, along with a method to iteratively estimate capillary oxygen concentrations. Pathological structural data for these simulations came from sections of the tumor stained with hematoxylin and eosin and were used to define the capillary positions and shapes, while overlapping regions of low oxygen concentration were defined by the hypoxia marker pimonidazole. These simulations were used to calculate spatial maps of the oxygen concentration and were tested for their ability to reproduce Eppendorf pO₂ histograms from the same tumor line. This simulation study predicted that capillary oxygen concentrations ranged from zero to above 20 μM, with a dominant peak in the hypoxic regions showing 78% of capillaries with less than 1 μM oxygen concentration, compared to only 12% in the non-hypoxic regions. The results were not highly sensitive to the metabolic oxygen consumption rate, within the range of 2 to 16 μM/s. This numerical method for oxygen capillary simulation is readily adaptable to histology sections and provides a method to examine the heterogeneity of oxygen within the capillaries and throughout the tumor tissue section being examined.

Sun, L-Q., Buchegger, F., Coucke, P. A. and Mirimanoff, R-O. Fractionated Irradiation Combined with Carbogen Breathing and Nicotinamide of Two Human Glioblastomas Grafted in Nude Mice.

This study addressed the potential radiosensitizing effect of nicotinamide and/or carbogen on human glioblastoma xenografts in nude mice. U-87MG and LN-Z308 tumors were irradiated with either 20 fractions over 12 days or 5 fractions over 5 days in air-breathing mice, mice injected with nicotinamide, mice breathing carbogen, or mice receiving nicotinamide plus carbogen. The responses to treatment were assessed using local control and moist desquamation. In U-87MG tumors, the enhancement ratios (ERs) at the radiation dose required to produce local tumor control in 50% of the treated mice (TCD₅₀) with nicotinamide and/or carbogen ranged from 1.13 to 1.24 for irradiation in 20 fractions over 12 days. In LN-Z308 tumors, the ERs at the TCD₅₀ with nicotinamide and/or carbogen ranged from 1.22 to 1.40 for irradiation in 5 fractions over 5 days and from 1.11 to 1.30 in 20 fractions over 12 days, respectively. Skin injury was slightly enhanced, with ERs ranged from 1.06 to 1.15 when radiation was combined with carbogen and/or nicotinamide. Thus carbogen and nicotinamide can slightly improve the radiation response of human glioblastoma xenografts.

Green, L. M., Murray, D. K., Tran, D. T., Bant, A. M., Kazarians, G., Moyers, M. F. and Nelson, G. A. Response of Thyroid Follicular Cells to Gamma Irradiation Compared to Proton Irradiation. I. Initial Characterization of DNA Damage, Micronucleus Formation, Apoptosis, Cell Survival, and Cell Cycle Phase Redistribution.

The RBE of protons has been assumed to be equivalent to that of photons. The objective of this study was to determine whether radiation-induced DNA and chromosome damage, apoptosis, cell killing and cell cycling in organized epithelial cells was influenced by radiation quality. Thyroid-stimulating hormone-dependent Fischer rat thyroid cells, established as follicles, were exposed to γ rays or proton beams delivered acutely over a range of physical doses. Gamma-irradiated cells were able to repair DNA damage relatively rapidly so that by 1 h postirradiation they had approximately 20% fewer exposed 3′ ends than their counterparts that had been irradiated with proton beams. The persistence of free ends of DNA in the samples irradiated with the proton beam implies that either more initial breaks or a quantitatively different type of damage had occurred. These results were further supported by an increased frequency of chromosomal damage as measured by the presence of micronuclei. Proton-beam irradiation induced micronuclei at a rate of 2.4% per gray, which at 12 Gy translated to 40% more micronuclei than in comparable γ-irradiated cultures. The higher rate of micronucleus formation and the presence of larger micronuclei in proton-irradiated cells was further evidence that a qualitatively more severe class of damage had been induced than was induced by γ rays. Differences in the type of damage produced were detected in the apoptosis assay, wherein a significant lag in the induction of apoptosis occurred after γ irradiation that did not occur with protons. The more immediate expression of apoptotic cells in the cultures irradiated with the proton beam suggests that the damage inflicted was more severe. Alternatively, the cell cycle checkpoint mechanisms required for recovery from such damage might not have been invoked. Differences based on radiation quality were also evident in the α components of cell survival curves (0.05 Gy⁻¹ for γ rays, 0.12 Gy⁻¹ for protons), which suggests that the higher level of survival of γ-irradiated cells could be attributed to the persistence of nonlethally irradiated thyrocytes and/or the capacity to repair damage more effectively than cells exposed to equal physical doses of protons. The final assessment in this study was radiation-induced cell cycle phase redistribution. Gamma rays and protons produced a similar dose-dependent redistribution toward a predominantly G₂-phase population. From our cumulative results, it seems likely that a majority of the proton-irradiated cells would not continue to divide. In conclusion, these findings suggest that there are quantitative and qualitative differences in the biological effects of proton beams and γ rays. These differences could be due to structured energy deposition from the tracks of primary protons and the associated high-LET secondary particles produced in the targets. The results suggest that a simple dose-equivalent approach to dosimetry may be inadequate to compare the biological responses of cells to photons and protons.

Zhou, P. K. and Rigaud, O. Down-Regulation of the Human CDC16 Gene after Exposure to Ionizing Radiation: A Possible Role in the Radioadaptive Response.

We have used the method of differential display of mRNAs to search for changes in gene expression associated with radioadaptation triggered by low doses of ionizing radiation in human lymphoblasts. We isolated a cDNA designated PB13 that was down-regulated as early as 1 h after irradiation with 4 Gy in cells adapted by a pre-exposure to a dose of 2 cGy, compared to 3 h in nonadapted cells (4 Gy alone). Northern analysis confirmed the differential expression of a 2.4-kb transcript that was repressed for at least 10 h after irradiation. The major part of the PB13 cDNA was identical to the human CDC16 mRNA. When using either PB13 or CDC16 cDNA as probes, similar radiation-induced alterations in gene expression were observed. Expression of the CDC16 gene was also repressed after oxidative stress with H₂O₂. The CDC16 protein belongs to the anaphase-promoting complex (APC) that controls progression through mitosis. The repression of expression of the CDC16 gene by ionizing radiation could result in delayed progression of damaged cells through mitosis. This cycle delay would occur earlier in adapted cells and would allow a more rapid and efficient repair that could contribute to the tolerance to subsequent irradiation.

Zölzer, F. and Streffer, C. G₂-Phase Delays after Irradiation and/or Heat Treatment as Assessed by Two-Parameter Flow Cytometry.

Similar to what has been observed after irradiation, the fraction of G₂-phase cells increases as a consequence of heat treatment. On the basis of cell cycle distributions alone, however, it is difficult to say whether the two results are related. In particular, comparison is complicated by the fact that the accompanying changes in the S-phase transition are different. These changes play a minor role after irradiation but constitute by far the most important cell cycle effect after heat treatment. Two-parameter flow cytometry was used here to study the proliferation of human melanoma cells in vitro. Cultures were pulse-labeled with BrdU after irradiation and/or heat treatment and were fixed either immediately or after a delay of up to 36 h. DNA-synthesizing cells were identified with the help of an FITC-conjugated antibody against BrdU; DNA was quantified after staining with propidium iodide. In this way, the cell cycle distribution could be determined and the progression through the cell cycle could be analyzed. From the movement of labeled cells through the cycle, in particular the appearance of labeled cells in the G₁ compartment (after they had gone through mitosis), the delay in G₂ phase could be determined. The duration of the G₂/M phase in control cells was about 6 h. This was increased to 12, 13 and 16 h after irradiation (4 Gy X rays), heat treatment (1 h at 43°C), and a combination of the two, respectively. In all these cases, the G₂-phase block was completely overcome within 48 h after treatment, whereas changes in the S phase were still observable at this time. As expected, the radiation-induced G₂-phase block was almost completely removed by incubating the cells with 5 or 10 mM caffeine. In the case of hyperthermia alone or in combination with radiation, however, caffeine was somewhat less effective. This does not mean, however, that the mechanisms involved are necessarily different. It can also be seen as a result of the differences in the time course of events. The long delay in S phase after heat treatment may lead to a loss of susceptibility to caffeine by the time the cells move into the G₂ phase.

Wu, H., George, K., Kawata, T., Willingham, V. and Cucinotta, F. A. Comparison of F Ratios Generated from Interphase and Metaphase Chromosome Damage Induced by High Doses of Low- and High-LET Radiation.

Although biophysical models predict a difference in the ratio of interchromosomal to intrachromosomal interarm exchanges (F ratio) for low- and high-LET radiations, few experimental data support this prediction. However, the F ratios in experiments to date have been generated using data on chromosome aberrations in samples collected at the first postirradiation mitosis, which may not be indicative of the aberrations formed in interphase after exposure to high-LET radiations. In the present study, we exposed human lymphocytes in vitro to 2 and 5 Gy of γ rays and 3 Gy of 1 GeV/nucleon iron ions (LET = 140 keV/μm), stimulated the cells to grow with phytohemagglutinin (PHA), and collected the condensed chromosomes after 48 h of incubation using both chemically induced premature chromosome condensation (PCC) and the conventional metaphase techniques. The PCC technique used here condenses chromosomes mostly in the G₂ phase of the cell cycle. The F ratio was calculated using data on asymmetrical chromosome aberrations in both the PCC and metaphase samples. It was found that the F ratios were similar for the samples irradiated with low- and high-LET radiation and collected at metaphase. However, for irradiated samples assayed by PCC, the F ratio was found to be 8.2 ± 2.0 for 5 Gy γ rays and 5.2 ± 0.9 for 3 Gy iron ions. The distribution of the aberrations indicated that, in the PCC samples irradiated with iron ions, most of the centric rings occurred in spreads containing five or more asymmetrical aberrations. These heavily damaged cells, which were either less likely to reach mitosis or may reach mitosis at a later time, were responsible for the difference in the F ratios generated from interphase and metaphase analysis after exposure to iron ions.

Ulsh, B. A., Whicker, F. W., Hinton, T. G., Congdon, J. D. and Bedford, J. S. Chromosome Translocations in T. scripta: The Dose-Rate Effect and In Vivo Lymphocyte Radiation Response.

Using a whole-chromosome FISH painting probe we previously developed for chromosome 1 of the yellow-bellied slider turtle (Trachemys scripta), we investigated the dose-rate effect for radiation-induced symmetrical translocations in T. scripta fibroblasts and lymphocytes. The dose rate below which no reduction in effect per unit dose is observed with further dose protraction was approximately 23 cGy h^–1. We estimated the whole-genome spontaneous background level of complete, apparently simple symmetrical translocations in T. scripta lymphocytes to be approximately 1.20 × 10^–3/cell projected from aberrations occurring in chromosome 1. Similar spontaneous background levels reported for humans are some 6- to 25-fold higher, ranging from about 6 × 10^–3 to 3.4 × 10^–2 per cell. This relatively low background level for turtles would be a significant advantage for resolution of effects at low doses and dose rates. We also chronically irradiated turtles over a range of doses from 0–8 Gy delivered at approximately 5.5 cGy h^–1 and constructed a lymphocyte dose–response curve for complete, apparently simple symmetrical translocations suitable for use with animals chronically exposed to radiation in contaminated environments. The best-fitting calibration curve (not constrained through the zero dose estimate) was of the form Y_as = c aD bD², where Y_as was the number of apparently simple symmetrical translocations per cell, D was the dose (Gy), a = (0.0058 ± 0.0009), b = (–0.00033 ± 0.00011), and c = (0.0015 ± 0.0013). With additional whole-chromosome probes to improve sensitivity, environmental biodosimetry using stable chromosome translocations could provide a practical and genetically relevant measurement end point for ecological risk assessments and biomonitoring programs.

Livshits, L. A., Malyarchuk, S. G., Lukyanova, E. M., Antipkin, Y. G., Arabskaya, L. P., Kravchenko, S. A., Matsuka, G. H., Petit, E., Giraudeau, F., Gourmelon, P., Vergnaud, G. and Le Guen, B. Children of Chernobyl Cleanup Workers do not Show Elevated Rates of Mutations in Minisatellite Alleles.

The disaster at the Chernobyl Nuclear Power Plant in April 1986 was accompanied by the release of large amounts of radioisotopes, resulting in the contamination of extensive regions of the Ukraine, Byelorus and the Russian Federation. Cleanup workers (liquidators) and people living on land contaminated with radioactive materials were most exposed. To assess the genetic effects of exposure to ionizing radiation after the Chernobyl accident, we have measured the frequency of inherited mutant alleles at seven hypermutable minisatellite loci in 183 children born to Chernobyl cleanup workers (liquidators) and 163 children born to control families living in nonirradiated areas of the Ukraine. There was no significant difference in the frequency of inherited mutant alleles between the exposed and control groups. The exposed group was then divided into two subgroups according to the time at which the children were conceived with respect to the fathers' work at the power plant. Eighty-eight children were conceived either while their fathers were working at the facility or up to 2 months later (Subgroup 1). The other 95 children were conceived at least 4 months after their fathers had stopped working at the Chernobyl site (Subgroup 2). The frequencies of mutant alleles were higher for the majority of loci (i.e. 1.44 times higher for CEB1) in Subgroup 1 than in Subgroup 2. This result, if confirmed, would reconcile the apparently conflicting results obtained in the chronically exposed Byelorus population and the Hiroshima-Nagasaki A-bomb survivors.

Kusunoki, Y., Hayashi, T., Morishita, Y., Yamaoka, M., Maki, M., Hakoda, M., Kodama, K., Bean, M. A. and Kyoizumi, S. T-Cell Responses to Mitogens in Atomic Bomb Survivors: A Decreased Capacity to Produce Interleukin 2 Characterizes the T Cells of Heavily Irradiated Individuals.

Significant decreases in the fraction of lymphocytes that are CD4 and increases in serum levels of some classes of immunoglobulin have been reported to occur in atomic bomb (A-bomb) survivors and in victims of the Chernobyl nuclear plant accident. To investigate the long-term effects of nuclear radiation on cellular immunity in more detail, we used limiting dilution assays with peripheral blood mononuclear cell preparations to analyze the T-cell responses of 251 A-bomb survivors exposed to less than 0.005 Gy and 159 survivors exposed to more than 1.5 Gy. The percentages of CD2-positive cells that were capable of proliferating in response to phytohemagglutinin (PHA) in the presence of exogenous interleukin 2 (IL2) did not differ substantially between distally exposed and more heavily exposed survivors. The heavily exposed survivors appeared to possess fewer T cells that were capable of proliferating in response to concanavalin A (Con A) or of producing interleukin 2. Assuming that CD4 T cells were the ones primarily responsible for producing IL2 in response to Con A, we were able to estimate how many cells in any given CD4 T-cell population were actually producing IL2. The results indicated that peripheral blood samples from heavily exposed survivors contained significantly fewer IL2-producing CD4 T cells than did similar samples from distally exposed survivors, indicating that significant exposure to A-bomb radiation may have a long-lasting negative effect on the capacity of CD4 T-cell populations to produce IL2.

Wilson, W. E., Lynch, D. J., Wei, K. and Braby, L. A. Microdosimetry of a 25 keV Electron Microbeam.

Electron microbeam experiments are planned or under way to explore in part the question regarding whether the bystander effect is a general phenomenon or is restricted to high-LET radiation. Since low-LET radiations scatter more readily compared to high-LET radiations, identifying bystander cells and assessing the potential dose that they may receive will be crucial to the interpretation of radiobiological results. This paper reports on initial calculations of the basic information needed for a stochastic model of the penetration of energetic electrons in tissue-like matter; the model will be used to predict doses delivered to adjacent regions in which bystander cells may reside. Results are presented of calculations of the stochastics of energy deposition by 25 keV electrons slowing down in a homogeneous water medium. Energy deposition distributions were scored for 1-μm spheres located at various penetration and radial distances up to 10 μm from the point of origin. The energy of 25 keV was selected because experiments are planned for that energy. At 25 keV there is a high probability that the entire electron track will be contained within a typical mammalian cell. Individual tracks are scored because of their primacy; data for higher doses can be obtained by convoluting single-track distributions. The event frequency decreases approximately exponentially after the first micrometer to 1% at about 8 μm of penetration. Radially, the 1% contour extends to 3.5 μm at a penetration of 5.5 μm. The frequency-mean energy deposited decreases from 1.5 to 1 keV/μm at a penetration of 3.5 μm, then increases back to about 1.5 at a penetration of 6.5 μm. The mean energy increases to about 3 keV/μm at a radial distance of 8.5 μm.

Hahn, F. F., Muggenburg, B. A., Snipes M. B. and Boecker, B. B. The Toxicity of Insoluble Cerium-144 Inhaled by Beagle Dogs: Non-neoplastic Effects.

The biological effects of inhaled β-particle-emitting radionuclides are not well known. The non-neoplastic diseases induced by an inhaled, relatively insoluble form of cerium-144 (¹⁴⁴Ce) were studied in beagle dogs exposed to graded activity levels of ¹⁴⁴Ce in fused aluminosilicate particles by a single, brief inhalation exposure and observed for their life span. The initial lung burdens (ILBs) achieved ranged from 0.000093–7.6 MBq ¹⁴⁴Ce/kg body weight. The ¹⁴⁴Ce was retained in the lung with an effective half-life of about 190 days. Significant ¹⁴⁴Ce was translocated to the tracheobronchial lymph nodes, and the concentration exceeded that of the lung at about 400 days after inhalation exposure. Significant radiation doses were delivered to the lung and tracheobronchial lymph nodes and to the heart adjacent to the tracheobronchial lymph nodes. Radiation pneumonitis was the predominant non-neoplastic disease. The dose response for radiation pneumonitis indicated that an ILB of 1.4 MBq/kg would cause death from radiation pneumonitis in 50% of the exposed dogs. This ILB resulted in a pulmonary dose to death of about 350 Gy. The tracheobronchial lymph nodes developed lesions in dogs with ILBs lower than those causing radiation pneumonitis. The overall results of this study, however, showed that ¹⁴⁴Ce, inhaled in an insoluble form, did not cause any unique or inexplicable biological effects in dogs or cause effects at unusually low doses that might call current radiation protection guidelines into question.

Vijayalaxmi, Pickard, W. F., Bisht, K. S., Leal, B. Z., Meltz, M. L., Roti Roti, J. L., Straube, W. L. and Moros, E. G. Cytogenetic Studies in Human Blood Lymphocytes Exposed In Vitro to Radiofrequency Radiation at a Cellular Telephone Frequency (835.62 MHz, FDMA).

Freshly collected peripheral blood samples from four healthy human volunteers were diluted with RPMI 1640 tissue culture medium and exposed in sterile T-75 tissue culture flasks in vitro for 24 h to 835.62 MHz radiofrequency (RF) radiation, a frequency employed for customer-to-base station transmission of cellular telephone communications. An analog signal was used, and the access technology was frequency division multiple access (FDMA, continuous wave). A nominal net forward power of 68 W was used, and the nominal power density at the center of the exposure flask was 860 W/m². The mean specific absorption rate in the exposure flask was 4.4 or 5.0 W/kg. Aliquots of diluted blood that were sham-exposed or exposed in vitro to an acute dose of 1.50 Gy of γ radiation were used as negative or positive controls. Immediately after the exposures, the lymphocytes were stimulated with a mitogen, phytohemagglutinin, and cultured for 48 or 72 h to determine the extent of genetic damage, as assessed from the frequencies of chromosomal aberrations and micronuclei. The extent of alteration in the kinetics of cell proliferation was determined from the mitotic indices in 48-h cultures and from the incidence of binucleate cells in 72-h cultures. The data indicated no significant differences between RF-radiation- and sham-exposed lymphocytes with respect to mitotic indices, incidence of exchange aberrations, excess fragments, binucleate cells, and micronuclei. In contrast, the response of the lymphocytes exposed to γ radiation was significantly different from both RF-radiation- and sham-exposed cells for all of these indices. Thus, under the experimental conditions tested, there is no evidence for the induction of chromosomal aberrations and micronuclei in human blood lymphocytes exposed in vitro for 24 h to 835.62 MHz RF radiation at SARs of 4.4 or 5.0 W/kg.

Kadhim, M. A., Marsden, S. J., Malcolmson, A. M., Folkard, M., Goodhead, D. T., Prise, K. M. and Michael, B. D. Long-Term Genomic Instability in Human Lymphocytes Induced by Single-Particle Irradiation.

Recent evidence suggests that genomic instability, which is an important step in carcinogenesis, may be important in the effectiveness of radiation as a carcinogen, particularly for high-LET radiations. Understanding the biological effects underpinning the risks associated with low doses of densely ionizing radiations is complicated in experimental systems by the Poisson distribution of particles that can be delivered. In this study, we report an approach to determine the effect of the lowest possible cellular radiation dose of densely ionizing α particles, that of a single particle traversal. Using microbeam technology and an approach for immobilizing human T-lymphocytes, we have measured the effects of single α-particle traversals on the surviving progeny of cells. A significant increase in the proportion of aberrant cells is observed 12–13 population doublings after exposure, with a high level of chromatid-type aberrations, indicative of an instability phenotype. These data suggest that instability may be important in situations where even a single particle traverses human cells.

Rhim, J. S. Molecular and Genetic Mechanisms of Prostate Cancer.

Prostate cancer is the most commonly diagnosed malignancy in American men and is the second leading cause of cancer death in males in the United States. Despite its high incidence, the molecular and genetic events involved in progression of prostate cancer remain poorly understood. In vitro models of human prostate epithelial (HPE) cells provide a practical approach to the analysis of the molecular and genetic mechanisms underlying prostate carcinogenesis. We reported the immortalization of normal adult HPE cells by transfection of the HPV-18 DNA and the subsequent conversion of such nontumorigenic but immortalized cells (HPV-18 C-1) into tumorigenic cells by the introduction of an activated Kras oncogene. Recently, we have demonstrated the malignant transformation of HPV-18 C-1 cells after multiple exposures to the chemical carcinogen N-nitroso-N-methylurea (NMU). Such transformants showed morphological alterations and anchorage-independent growth in soft agar and induced carcinomas when transplanted into nude mice. No TP53 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 (p10>ptr) (the most frequent cytogenetic changes observed directly in prostate cancer patients) were observed only in the tumor outgrowths. These findings provide the first evidence of malignant transformation of HPE cells exposed to a chemical carcinogen.

Maitland, N. J., Macintosh, C. A., Hall, J., Sharrard, M., Quinn, S. and Lang, S. In Vitro Models to Study Cellular Differentiation and Function in Human Prostate Cancers.

To augment the currently available models of human prostate cancer in vitro, we have established extended life-span epithelial cultures from biopsies of well-differentiated prostate cancers. The genetic identity of the target cells was assessed by allelotyping, using microsatellites located on chromosome 8p, and microdissection of tissues and primary cell cultures. Cells with an extended life span (PxE6) were derived by recombinant retrovirus infection to introduce the human papilloma virus E6 gene (epithelial cells). Immunophenotyping of the resultant cell strains confirmed retention of differentiated cell functions, and the genotype of the E6-expressing epithelial cells was stable, while SV40-immortalized cultures were more unstable, leading to tetraploidy. All PxE6 cells eventually senesced, but an immortalized epithelial culture, P4E6, was derived from one of the epithelial cultures. The properties of this cell line, which remains close to diploid, are similar to those of early prostate cancer cells, and it retains expression of many prostate-associated antigens, such as prostate-specific antigen (PSA).

Ratsch, S. B., Gao, Q., Srinivasan, S., Wazer, D. E. and Band, V. Multiple Genetic Changes Are Required for Efficient Immortalization of Different Subtypes of Normal Human Mammary Epithelial Cells.

Breast cancer is the second leading cause of cancer-related deaths of women in the U.S. About 180,000 new cases of breast cancer are diagnosed each year, a quarter of them fatal. Early detection is the key to the survival of these patients. However, there are no molecular markers to detect breast cancer at very early stages. A hurdle in understanding the early molecular changes in breast cancer has been the difficulty in establishing premalignant lesions and primary breast tumors as in vitro cell cultures. Normal epithelial cells grow for a finite life span and then senesce. Immortalization is defined by continuous growth of otherwise senescing cells and is believed to represent an early stage in tumor progression. To examine these early stages, we and others have developed in vitro models of mammary epithelial cell immortalization. These models have been extremely important in understanding the role of various tumor suppressor pathways that maintain the normal phenotypes of mammary epithelial cells. In this paper, we describe the establishment of these models and their relevance to understanding the molecular changes that occur in early breast cancer. These models have helped to identify molecular changes that occur in early breast cancers and appear to be well suited to identify novel markers for early diagnosis of breast cancer.

Russo, J. and Russo, I. H. The Pathway of Neoplastic Transformation of Human Breast Epithelial Cells.

The morphological analysis of breast cancer development indicates this to be a multistep process that progressively evolves from ductal hyperplasia and atypical ductal hyperplasia, which represent the initial stages of neoplastic growth, to carcinoma in situ, invasive carcinoma, and ultimately metastasis, as has been documented for a number of other malignancies. The understanding of the cellular and molecular processes that lead a normal cell to malignancy requires the analysis of pure populations of human breast epithelial cells (HBEC) representing specific stages of neoplastic progression. The neoplastic transformation of HBEC in vitro represents a successful model for obtaining knowledge about the molecular and biological alterations that may contribute to the tumorigenic mechanisms. We present here a current understanding of chemically transformed HBEC in the following aspects: (1) factors affecting the transformation of HBEC such as immortalization; (2) new targets for studying the mechanism of cell immortalization such as alterations in telomerase activity, differential expression of cell cycle-dependent genes, and others recently isolated through differential cloning, such as H-ferritin, and a calcium binding protein; (3) genetic mechanisms underlying cell transformation; and (4) application of the microcell-mediated chromosome transfer technique as an approach to testing the functional role of specific genes whose dysregulation or loss of function may contribute to the ultimate cell transformation. Further efforts in this cell system will be directed to determining the roles of identified molecular changes as well as the mapping/cloning of tumor suppressor or senescence genes.

Solomon, C., White, J. H., Rhim, J. S. and Kremer R. Vitamin D Resistance in RAS-Transformed Keratinocytes: Mechanism and Reversal Strategies.

Human retinoid X receptor α (hRXRα) plays a critical role in DNA binding and transcriptional activity through its heterodimeric association with several members of the nuclear receptor superfamily, including the vitamin D receptor (VDR). Several cancer cell lines derived from different tissues have been shown to be resistant to the growth-inhibitory action of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], the biologically active metabolite of vitamin D₃. Here we show that in RAS-transformed keratinocytes, Ser260 of hRXRα is phosphorylated through the RAS-RAF-MAP kinase cascade. This phosphorylation event results in the inhibition of vitamin D signaling via VDR/hRXRα heterodimers. Strategies to reverse this resistance include the use of the MAP kinase inhibitor, PD098059, and a non-phosphorylatable hRXRα mutant, Ala260, which completely abolishes RXR phosphorylation and restores the function of both 1,25(OH)₂D₃ and a specific RXR ligand, LG1069 (4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphtalenyl)ethenyl]-benzoic acid). In addition, we show that a vitamin D analog with low calcemic activity (EB1089) is more potent than 1,25(OH)₂D₃ in inhibiting cancer cell growth in this system. Targeted therapy with selective analogs such as EB1089, in combination with the inhibition of phosphorylation of the RXR, could play a critical role in the development of strategies for cancer treatment.

Miller, A. C., Xu, J., Stewart, M. and McClain, D. Suppression of Depleted Uranium-Induced Neoplastic Transformation of Human Cells by the Phenyl Fatty Acid, Phenyl Acetate: Chemoprevention by Targeting the p21RAS Protein Pathway.

Depleted uranium is a dense heavy metal used primarily in military applications. Published data from our laboratory have demonstrated that exposure to depleted uranium in vitro can transform immortalized human osteoblast (HOS) cells to the tumorigenic phenotype (associated with aberrant RAS oncogene expression and tumor suppressor protein production). Since depleted uranium is used in military applications, it would therefore be beneficial to identify and test potential antitumor-promoting agents. Chemopreventive interventions that target deregulated signal transduction pathways may be effective strategies to prevent carcinogenesis. Since the RAS protein plays a key role in signal transduction, disruption of its signaling pathway may be particularly effective. The phenyl fatty acid, phenyl acetate, a differentiation inducer that affects post-translational processing of RAS, was tested for its ability to prevent depleted uranium-induced neoplastic transformation using HOS cells. After a 24-h exposure to insoluble depleted uranium–uranium dioxide (1 mg/ml), cells were incubated for 1 day to 6 weeks with 2.5 mM phenyl acetate. Treatment with depleted uranium resulted in transformation to the tumorigenic phenotype. In contrast, HOS cells exposed to depleted uranium and then treated with phenyl acetate did not exhibit transformation to the tumorigenic phenotype. These data suggest that depleted uranium-induced neoplastic transformation in vitro can be prevented by targeting the RAS protein.

Newton, D. L., Kaur, G., Rhim, J. S., Sausville, E. A. and Rybak, S. M. RNA Damage and Inhibition of Neoplastic Endothelial Cell Growth: Effects of Human and Amphibian Ribonucleases.

Angiogenesis defines the many steps involved in the growth and migration of endothelial cell-derived blood vessels. This process is necessary for the growth and metastasis of tumors, and considerable effort is being expended to find inhibitors of tumor angiogenesis. This usually involves screening of potential anti-angiogenic compounds on endothelial cells. To this end, two candidate anti-angiogenic RNA-damaging agents, onconase and (-4)rhEDN, were screened for their effects on endothelial cell proliferation using three distinct types of endothelial cells in culture: HPV-16 E6/E7-immortalized human umbilical vein endothelial cells (HUVECs), a Kras-transformed HPV-16 E6/E7 HUVEC (Rhim et al., Carcinogenesis 4, 673–681, 1998), and primary HUVECs. Onconase similarly inhibited proliferation in all three cell lines (IC₅₀ = 0.3–1.0 μM) while (-4)rhEDN was more effective on immortalized HUVEC cell lines (IC₅₀ = 0.02–0.06 μM) than on primary HUVECs (IC₅₀ > 0.1 μM). Differential sensitivity to these agents implies that more than one endothelial cell type must be used in proliferation assays to screen for novel anti-angiogenic compounds.

Trosko, J. E. and Chang, C. C. Role of Stem Cells and Gap Junctional Intercellular Communication in Human Carcinogenesis.

Epidemiological data, experimental animal bioassays, studies of in vitro neoplastic transformation, and molecular oncology studies have implicated a multistage, multimechanism process in human carcinogenesis. From animal carcinogenesis studies, the operational concept of a single normal cell being irreversibly altered during the first step in carcinogenesis is called initiation. The subsequent interruptible or reversible clonal expansion of these initiated cells by non-cytotoxic mitogenic stimuli, compensatory hyperplasia due to cell death by necrosis, or inhibition of apoptosis is referred to as the promotion phase. Last, when one of these clonally expanded, initiated cells acquires sufficient genetic/epigenetic alterations to become neoplastically transformed and acquire the phenotypes of promoter independence, invasiveness and metastasis, it is referred to as the progression step of carcinogenesis. This report hypothesizes that the single normal cell that is initiated is a pluripotent stem cell. By assuming that the normal pluripotent stem cell is immortal and becomes mortal when induced to terminally differentiate, initiation would be viewed as the irreversible process by which a stable alteration in a finite number of proto-oncogenes and/or tumor suppressor genes could block terminal differentiation or “mortalization”. Promotion would involve the reversible inhibition of gap junctional intercellular communication (GJIC) and while progression occurs with the stable down-regulation of GJIC.

Rouzaut, A., Recio, J. A. and Notario, V. Expression of the Protein Product of the PCPH Proto-oncogene in Human Tumor Cell Lines.

Exposure of Syrian hamster embryo fibroblasts to chemical carcinogens resulted in the oncogenic activation of the PCPH proto-oncogene by induction of a single base-pair deletion that generated a truncated PCPH oncoprotein (mutated PCPH). Recently, we isolated and characterized the cDNA for the human PCPH proto-oncogene and determined that in humans PCPH is a single-copy gene located in chromosome 14 (14q24.3). Pilot mRNA expression studies indicated that PCPH was expressed in the majority of normal organs tested, particularly in liver and kidney, but it appeared to be expressed either at low levels or not at all in tumor cells or cell lines derived from the high-expressing tissues. We have generated an antiserum against bacterial recombinant Syrian hamster PCPH. This antiserum recognizes both the normal and truncated, oncogenic Syrian hamster PCPH proteins and cross-reacts with the yeast, mouse, rat and human homologue proteins. Using this antibody, we have performed a study of PCPH expression in a larger sample of human neoplastic cell lines, including some derived from breast, nervous system, colon, lung and pancreas tumors. Results confirmed the frequent lack of PCPH expression in malignant cells and identified several immunoreactive forms of PCPH being differentially expressed in cells of diverse tissue origins.

Shay, J. W. and Wright, W. E. Telomeres and Telomerase: Implications for Cancer and Aging.

Maintenance of telomere stability is required for cells to escape from replicative senescence and proliferate indefinitely. Telomere length is maintained by a balance between processes that lengthen telomeres (telomerase) and processes that shorten telomeres (the end-replication problem). Telomerase is a cellular ribonucleoprotein reverse transcriptase which stabilizes telomere length by adding hexameric (TTAGGG) repeats to the telomeric ends of the chromosomes, thus compensating for the continued erosion of telomeres. Introduction of the telomerase catalytic protein component into normal telomerase-negative human cells results in restoration of telomerase activity and extension of cellular life span. Human cells with introduced telomerase maintain a normal chromosome complement and continue to grow in a normal manner. Telomerase-induced manipulations of telomere length may thus be important not only for cell and tissue engineering but also for dissecting the molecular mechanisms underlying inherited genetic diseases, as well as defining the genetic pathways leading to cancer. Because almost all human tumors express telomerase activity, inhibition of telomerase may result in gradual erosion of telomeres and eventual cessation of cell proliferation or induction of apoptosis. Thus telomerase may also be a promising target for cancer therapy.

Reddel, R. R., Bryan, T. M., Colgin, L. M., Perrem, K. T. and Yeager, T. R. Alternative Lengthening of Telomeres in Human Cells.

Activation of a telomere maintenance mechanism appears to be essential for immortalization. In most human tumors and tumor cell lines, the telomere maintenance mechanism involves the activity of telomerase, a reverse transcriptase holoenzyme that synthesizes telomeric repeat DNA. In some cases, telomere maintenance occurs in the absence of telomerase activity by a mechanism referred to as alternative lengthening of telomeres (ALT). The development of telomere-targeted anticancer therapies will be facilitated by an understanding of the molecular mechanisms of ALT and of the means whereby ALT is repressed in normal cells.

Chang, C. C., Sun, W., Saitoh, M., Tai, M-H. and Trosko, J. E. A Human Breast Epithelial Cell Type with Stem Cell Characteristics as Target Cells for Carcinogenesis.

Two types of human breast epithelial cells (HBEC) have been characterized. In contrast to Type II HBEC, which express basal epithelial cell phenotypes, Type I HBEC are deficient in gap junctional intercellular communication and are capable of anchorage-independent growth and of expressing luminal epithelial cell markers, estrogen receptors, and stem cell characteristics (i.e. the ability to differentiate into other cell types and to form budding/ductal organoids on Matrigel). A comparative study of these two types of cells has revealed a high susceptibility of Type I HBEC to immortalization by SV40 large T antigen, although both types of cells are equally capable of acquiring an extended life span (bypassing senescence) after transfection with SV40. The immortalization was accompanied by elevation of a low level of telomerase activity in the parental cells after mid-passage (∼60 cumulative population doubling levels). Thus HBEC do have a low level of telomerase activity, and Type I HBEC with stem cell characteristics are more susceptible to telomerase activation and immortalization, a mechanism which might qualify them as target cells for breast carcinogenesis. The immortalized Type I HBEC can be converted to highly tumorigenic cells by further treatment with X rays (2 Gy × 2) and transfection with a mutated ERBB2 (also known as NEU) oncogene, resulting in the expression of p185^ERBB2 which is tyrosine phosphorylated.

Sakaguchi, M., Yamada, H., Tsuji, T., Inoue, Y., Miyazaki, M., Tanaka, T. and Namba, M. Loss of Nuclear Localization of the S100C Protein in Immortalized Human Fibroblasts.

It is well known that cancer develops through a multistep process. In vitro transformation studies of normal human cells have shown that the immortalization step is critical for neoplastic transformation of cells. Furthermore, studies of cell fusion between normal and immortalized cells have indicated that the normal phenotype is dominant and the immortal phenotype is recessive. Thus we looked for cellular proteins that were down-regulated in immortalized human cells by two-dimensional gel electrophoresis to elucidate the mechanisms of immortalization of human cells. We found that the S100C protein was down-regulated in immortalized cells. This protein was localized in the cytoplasm of cells at the semiconfluent stage, while at the confluent stage it moved into the nuclei of normal cells but not into those of immortalized cells. Microinjection of an S100C antibody into normal confluent cells diminished the level of nuclear S100C protein, resulting in DNA synthesis. Taken together, loss of nuclear localization of the S100C protein, which may be related to DNA synthesis, is thought to be one of the mechanisms of immortalization.

Syljuåsen, R. G., Krolewski, B. and Little, J. B. Molecular Events in Radiation Transformation.

Studies of human tumor cell lines have revealed alterations in the regulation of a number of cell cycle-related genes, associated in some cases with a TP53-independent loss of the radiation-induced G₁-phase arrest. It is not clear, however, whether these are early or late events in tumor development, or they arise in tumor cell lines during growth in culture. Since the oncogenic transformation of an individual cell is thought to be an early event in tumor development, we have used a model system of normal and radiation-transformed C3H 10T½ mouse fibroblast cell clones to address this issue. Transformed clones derived from type III foci were compared with clones derived from parental, wild-type cells. Approximately 25% of transformed clones showed Trp53 mutations in exon 5; however, preliminary results based on in situ immunofluorescence studies with an antibody recognizing mutant Trp53 indicate that the appearance of such mutations in transformed clones occurs late in the process of transformation and is unlikely to represent an initiating event. The remaining transformed clones and all clones derived from parental cells expressed wild-type Trp53. Radiation-induced G₁-phase arrest was either absent or significantly reduced in all of the transformed clones, independent of Trp53 status. Constitutive expression of Cdkn1a protein was significantly increased in most of the transformed clones. Also, the majority of transformed clones showed elevated levels of cyclin D1, and two clones overexpressed cyclin E. These results indicate that loss of G₁-phase checkpoint control, independent of Trp53 status, and altered expression of cell cycle regulatory proteins may represent early events in the process of radiation-induced carcinogenesis that are associated with the malignant transformation of individual cells.

Riches, A., Peddie, C., Rendell, S., Bryant, P., Zitzelsberger, H., Bruch, J, Smida, J, Hieber, L. and Bauchinger, M. Neoplastic Transformation and Cytogenetic Changes after Gamma Irradiation of Human Epithelial Cells Expressing Telomerase.

Neoplastic transformation of human epithelial cells by radiation has previously been investigated using cell lines immortalized with viral vectors. There are disadvantages to this approach, and we report here the results of studies using a human retinal pigment epithelial cell line (340RPE-T53) immortalized by treatment with telomerase. After exposure of the cells to fractionated doses of γ radiation, there was a marked increase in anchorage-independent growth of the surviving cells. The cloned cell lines derived from these anchorage-independent cultures exhibited an increased growth rate in vitro and were serum-independent compared with the parent cell line. The parent cell line maintained a stable diploid karyotype. The cell lines cloned after irradiation with the lower doses (10 × 2 Gy) were hypodiploid with loss of chromosome 13 and a high level amplification of 10p11.2 associated with a deletion of the remaining short arm segment of chromosome 10 distal to 10p11.2. In contrast, the cell lines cloned after irradiation with the higher doses (15 × 2 Gy) were near-tetraploid with derivative chromosomes present characterized by SKY analysis. Thus this human epithelial cell line immortalized with telomerase provides an improved model to investigate mechanisms of radiation carcinogenesis.

Zhao, Y. L., Piao, C. Q., Hall, E. J. and Hei, T. K. Mechanisms of Radiation-Induced Neoplastic Transformation of Human Bronchial Epithelial Cells.

Carcinogenesis is a multistage process with sequences of genetic events that govern the phenotypic expression of a series of transformation steps that lead to the development of metastatic cancer. To better understand the mechanisms involved in human bronchial carcinogenesis induced by α particles from radon, we have developed a model of neoplastic transformation based on human papillomavirus-immortalized human bronchial epithelial (BEP2D) cells. Cells exposed to α particles become tumorigenic after progressing through a series of sequential stages including altered growth pattern, resistance to serum-induced terminal differentiation, agar-positive growth, tumorigenicity, and metastasis, with each step representing a necessary yet insufficient step toward the later, more malignant phase. Cell fusion studies indicated that the radiation-induced tumorigenic phenotype in BEP2D cells can be completely suppressed by fusion with nontumorigenic BEP2D cells. Several cellular differentiation and growth regulation genes such as DCC (deleted in colorectal cancer), CDKN1A (also known as p21^C1P1) and the gene that encodes DNA-PK were frequently found to be modulated in tumorigenic BEP2D cells and may be related to the process of carcinogenesis.

Lechner, J. F., Fugaro, J. M., Wong, Y., Pass, H. I., Harris, C. C. and Belinsky, S. A. Perspective: Cell Differentiation Theory May Advance Early Detection of and Therapy for Lung Cancer.

Worldwide, 1,000,000 people succumb to lung cancer annually. Many of these deaths could be prevented if there were better screening methods to uncover the disease when it is limited and most responsive to intervention. Novel biomarkers of early-stage disease are therefore needed. By applying the principle of “oncology recapitulates ontogeny”, we have discovered three homeobox (HOX) genes that are inappropriately expressed in the majority of lung tumors. Understanding the role of these inappropriately expressed genes in lung epithelial cell carcinogenesis may not only augment early detection, but may also offer new avenues of treatment of this disease.

Roti Roti, J. L., Malyapa, R. S., Bisht, K. S., Ahern, E. W., Moros, E. G., Pickard, W. F. and Straube, W. L. Neoplastic Transformation in C3H 10T½ Cells after Exposure to 835.62 MHz FDMA and 847.74 MHz CDMA Radiations.

The effect of radiofrequency (RF) radiation in the cellular phone communication range (835.62 MHz frequency division multiple access, FDMA; 847.74 MHz code division multiple access, CDMA) on neoplastic transformation frequency was measured using the in vitro C3H 10T½ cell transformation assay system. To determine if 835.62 MHz FDMA or 847.74 MHz CDMA radiations have any genotoxic effects that induce neoplastic transformation, C3H 10T½ cells were exposed at 37°C to either of the above radiations [each at a specific absorption rate (SAR) of 0.6 W/kg] or sham-exposed at the same time for 7 days. After the culture medium was changed, the cultures were transferred to incubators and refed with fresh growth medium every 7 days. After 42 days, the cells were fixed and stained with Giemsa, and transformed foci were scored. To determine if exposure to 835.62 MHz FDMA or 847.74 MHz CDMA radiation has any epigenetic effects that can promote neoplastic transformation, cells were first exposed to 4.5 Gy of X rays to induce the transformation process and then exposed to the above radiations (SAR = 0.6 W/kg) in temperature-controlled irradiators with weekly refeeding for 42 days. After both the 7-day RF exposure and the 42-day RF exposure after X irradiation, no statistically significant differences in the transformation frequencies were observed between incubator controls, the sham-exposed (maintained in irradiators without power to the antenna), and the 835.62 MHz FDMA or 847.74 MHz CDMA-exposed groups.

Suzuki, K., Mori, I., Nakayama, Y., Miyakoda, M., Kodama, S. and Watanabe, M. Radiation-Induced Senescence-like Growth Arrest Requires TP53 Function but not Telomere Shortening.

Normal human diploid cells irradiated with X rays showed permanent cell cycle arrest and exhibited senescence-like phenotypes including the expression of senescence-associated β-galactosidase (SA-β-gal). X irradiation caused persistent phosphorylation of TP53 at Ser 15 and accumulation of the TP53 protein, followed by the induction of CDKN1A (also known as p21^Waf1/Cip1) and CDKN2A (also known as p16), preceded the expression of SA-β-gal. NCI-H1299 human lung carcinoma cells, in which no TP53 protein was expressed, were irradiated with X rays with or without the exogenous expression of TP53 gene. Although induction of TP53 protein alone could induce SA-β-gal expression, the frequency of SA-β-gal-positive cells was significantly increased when TP53-induced H1299 cells were exposed to X rays. The mean terminal restriction fragment length in normal human cells was approximately 12 kb and did not change in SA-β-gal-positive cells. These results indicate that ionizing radiation induces senescence-like growth arrest that is dependent on TP53 function but independent of telomere shortening. Our findings suggest that cells harboring irreparable DNA damage are programmed to undergo premature senescence to maintain the integrity of the genome.

Kodama, S., Mori, I., Roy, K., Yang, Z., Suzuki, K. and Watanabe, M. Culture Condition-Dependent Senescence-Like Growth Arrest and Immortalization in Rodent Embryo Cells.

We investigated the telomerase activity, telomere length, and replicative life span of cells from human embryos and rodent embryos (mouse, rat and Syrian hamster). We used two culture conditions for rodent embryo cells whereby the cells were plated at a density of 2 × 10⁵ into a 25-cm² flask and subcultured every 3 days or every 10 days. We found that nearly 100% of the cultures of rodent embryo cells become immortal when they are subcultured using the 10-day culture protocol. These rodent embryo cells retain telomerase activity and long telomeres (19–50 kb) in the long-term cultures, whereas human embryo cells rapidly deplete telomerase activity associated with significant shortening of telomeres, and then they senesce. In contrast to the results from 10-day cultures, we found that some mouse cell cultures and most Syrian hamster cell cultures arrest cell growth after 13 and 29 population doublings, respectively, while retaining substantial levels of telomerase activity and experiencing no significant loss of telomeres when the cells were subcultured using the 3-day culture protocol. This growth arrest is phenotypically indistinguishable from cellular senescence. The present results suggest that in rodent cells the onset of senescence-like arrest can be activated without repression of telomerase, and that this activation pathway can be bypassed easily under certain culture conditions, such as the 10-day culture protocol.

Piao, C. Q. and Hei, T. K. Gene Amplification and Microsatellite Instability Induced in Tumorigenic Human Bronchial Epithelial Cells by Alpha Particles and Heavy Ions.

Gene amplification and microsatellite alteration are useful markers of genomic instability in tumor and transformed cell lines. It has been suggested that genomic instability contributes to the progression of tumorigenesis by accumulating genetic changes. In this study, amplification of the carbamyl-P-synthetase, aspartate transcarbamylase, dihydro-orotase (CAD) gene in transformed and tumorigenic human bronchial epithelial (BEP2D) cells induced by either α particles or ⁵⁶Fe ions was assessed by measuring resistance to N-(phosphonacetyl)-l-aspartate (PALA). In addition, alterations of microsatellite loci located on chromosomes 3p and 18q were analyzed in a series of primary and secondary tumor cell lines generated in nude mice. The frequency of PALA-resistant colonies was 1–3 × 10^–3 in tumor cell lines, 5–8 × 10^–5 in transformed cells prior to inoculation into nude mice, and less than 10^–7 in control BEP2D cells. Microsatellite alterations were detected in all 11 tumor cell lines examined at the following loci: D18S34, D18S363, D18S877, D3S1038 and D3S1607. No significant difference in either PALA resistance or microsatellite instability was found in tumor cell lines that were induced by α particles compared to those induced by ⁵⁶Fe ions.