Fluorescent light induces malignant transformation in mouse embryo cell cultures.

Fluorescent light induced a dose-dependent malignant transformation in mouse C3H10T1/2 cells. A plateau in the dose-response curve for transformation was correlated with that observed with ultraviolet light exposure. The similarity in the two dose-response patterns suggests that similar molecular processes may be involved in the induction of malignant transformation by the two types of radiation.

Lung cancer induced in hamsters by low doses of alpha radiation from polonium-210.

Lung cancers have been induced in 9 to 53 percent of hamsters given multiple intratracheal instillations of polonium-210 in amounts yielding lifetime exposures of 15 to 300 rads to the lungs. Cigarette smokers have previously been estimated to receive 20 rads to areas of the bronchial epithelium from deposited polonium-210. This finding thus supports the hypothesis that alpha radiation resulting from the polonium-210 or lead-210 present in cigarette smoke may be a significant causative factor in human lung cancer.

X rays induce interallelic homologous recombination at the human thymidine kinase gene.

We have developed a human lymphoblast cell line for the study of interchromosomal homologous recombination at the endogenous thymidine kinase (tk) gene on chromosome 17 (M. B. Benjamin, H. Potter, D. W. Yandell, and J. B. Little, Proc. Natl. Acad. Sci. USA 88:6652-6656, 1991). This cell line (designated 6:86) carries unique heterozygous frameshift mutations in exons 4 and 7 of its endogenous tk alleles and can revert to TK+ by frame-restoring mutations, gene conversion, or reciprocal recombination. Line 6:86 reverts spontaneously to TK+ at a frequency of 10(-7) to 10(-8), and exposures to X-irradiation or the frameshift mutagen ICR-191 induce increased reversion frequencies in a dose-dependent manner. Another cell line (designated 4:2) carries a homozygous exon 7 frameshift and is not expected to revert through mechanisms other than frame-restoring mutation. Line 4:2 reverts to TK+ at a lower spontaneous frequency than does 6:86 but can be induced with similar kinetics by ICR-191. In contrast to line 6:86, however, X rays did not induce detectable reversion of line 4:2. We have characterized a number of 6:86-derived revertants by means of restriction fragment length polymorphism analysis at tk and linked loci, single-strand conformation polymorphisms, and direct transcript sequencing. For X rays, most revertants retain both original mutations in the genomic DNA, and a subset of these frameshift-retaining revertants produce frameshift-free message, indicating that reversion is the result of reciprocal recombination within the tk gene. Frame-restoring point mutations, restoration of original sequences, and phenocopy reversion by acquisition of aminopterin resistance were also found among X-ray-induced revertants, whereas the ICR-191-induced revertants examined show only loss of the exon 7 frameshift.

Elevated frequency of microsatellite mutations in TK6 human lymphoblast clones selected for mutations at the thymidine kinase locus.

A major question in carcinogenesis is, How can a normal cell accumulate multiple mutations in different genes on different chromosomes, when the mutation rate of each gene is in the range of 10(-8) to 10(-5) per cell division? We hypothesize that many mutations may not be isolated events but rather are accompanied by concomitant mutations elsewhere in the genome. To test this hypothesis, 331 independent clones selected for new mutations at the thymidine kinase (TK) locus on chromosome 17q, and 243 nonselected control clones were examined for mutations in 12 random microsatellite loci dispersed throughout the genome. A total of 24 second-site mutations were identified in the TK mutant clones, compared with 3 in the control clones not selected for mutations at TK. The mutations include small deletions, insertions, and loss of heterozygosity. These results provide evidence that a global trans-acting mutagenic process exists in human cells. The activation of this process could be responsible for causing multiple essential mutations in tumor cells.

[Nontargeted effects of ionizing radiation: implications for low-dose exposures].

The traditional thinking has been that the biological effects of ionizing radiation occur in irradiated cells as a consequence of the DNA damage they incur. This implies that: 1) biological effects occur only in irratiated cells, 2) radiation traversal through the nucleus of the cell is a prerequisite to produce a biological response, and 3) DNA is the target molecule in the cell. Evidence has been emerging, however, for non-DNA targeted effects of radiation; that is, effects including mutations, chromosomal aberrations, and changes in gene expression which occur in cells that in themselves receive no radiation exposure. Two of these phenomena will be described in this paper. The first is radiation-induced genomic instability whereby biological effects, including elevated frequencies of mutations and chromosomal aberrations, arise in the distant descendants of irradiated cells. The second phenomenon has been termed the "bystander effect", whereby in a mixed population of irradiated and nonirradiated cells, biological effects arise in those cells that receive no radiation exposure. The damage signals are transmitted from cell to cell through gap junction channels, and the genetic effects observed in bystander cells appear to result from an upregulation of oxidative stress. The possible influence of these non-targeted effects of radiation of the respounse to low-dose exposures is discussed.

gamma-H2AX foci after low-dose-rate irradiation reveal atm haploinsufficiency in mice.

We have investigated the use of the gamma-H2AX assay, reflecting the presence of DNA double-strand breaks (DSBs), as a possible means for identifying individuals who may be intermediate with respect to the extremes of hyper-radiosensitivity phenotypes. In this case, cells were studied from mice that were normal (Atm+/+), heterozygous (Atm+/-), or homozygous recessive (Atm-/-) for a truncating mutation in the Atm gene. After single acute (high-dose-rate) exposures, differences in mean numbers of gamma-H2AX foci per cell between samples from Atm+/+ and Atm-/- mice were clear at nearly all sampling times, but at no sampling time was there a clear distinction for cells from Atm+/+ and Atm+/- mice. In contrast, under conditions of low-dose-rate irradiation at 10 cGy/h, appreciable differences in the levels of gamma-H2AX foci per cell were observed in synchronized G1 cells derived from Atm+/- mice relative to cells from Atm+/+ mice. The levels were intermediate between those for cells from Atm+/+ and Atm-/- mice. After 24 h exposure at this dose rate, measurements in cells from four different mice for each genotype yielded mean frequencies of foci per cell of 1.77 +/- 0.13 (SEM) for Atm+/+ cells, 4.75 +/- 0.20 for the Atm+/- cells, and 11.10 +/- 0.33 for the Atm-/-cells. The distributions of foci per G1 cell were not significantly different from Poisson. To the extent that variations in sensitivity with respect to gamma-H2AX focus formation reflect variations in radiosensitivity for biological effects of concern, such as carcinogenesis, and that similar differences are seen for other genetic DNA DSB processing defects in general, this assay may provide a relatively straightforward means for distinguishing individuals who may be mildly hypersensitive to radiation such as we observed for Atm heterozygous mice.

Levels of gamma-H2AX Foci after low-dose-rate irradiation reveal a DNA DSB rejoining defect in cells from human ATM heterozygotes in two at families and in another apparently normal individual.

We have investigated the use of the gamma-H2AX assay, reflecting the presence of DNA double-strand breaks, as a possible means for identifying individuals who are mildly hypersensitive to ionizing radiation, such as some ATM heterozygotes. We compared levels of gamma-H2AX foci after irradiation in cells from six apparently normal individuals as well as from individuals from two separate AT families including the proband, mother, father and three unaffected siblings in each family. After a 1-Gy single acute (high-dose-rate) gamma-ray dose delivered to noncycling contact-inhibited monolayers of cells, clear differences were seen between samples from normal individuals (ATM(+/+)) and probands (ATM(-/-)) at nearly all sampling times after irradiation, but no clear distinctions were seen for cells from normal compared to obligate heterozygotes (ATM(+/-)). In contrast, after 24 h of continuous irradiation at a dose rate of 10 cGy/h, appreciable differences in numbers of foci per cell were observed for cells from individuals for all the known ATM genotypes compared with controls. Four unaffected siblings had mean numbers of foci per cell similar to that for the obligate heterozygotes, whereas the other two had mean values similar to that for normal controls. We determined independently that those siblings with mean numbers of foci per cell in the range of ATM heterozygotes carried the mutant allele, while both siblings with a normal number of foci per cell after irradiation had normal alleles. A more limited set of experiments using lymphoblastoid cell strains in the low-dose-rate assay also revealed distinct differences for normal compared to ATM heterozygotes from the same families and opens the possibility of using peripheral blood lymphocytes as a more suitable material for an assay to detect mild hypersensitivities to radiation among individuals.

Establishment and characteristics of a hamster lung adenocarcinoma in vivo and in vitro.

Several cell lines designated HLAC were derived from primary lung carcinomas induced in Syrian hamsters by polonium-210 or benzo[a]pyrene. Primary tumor nodules were initially transplanted into cheek pouches, and the tumors that grew were passed into tissue culture and into the cheek pouches of other hamsters for continued in vivo passage. By serial passage and cloning, cell lines were isolated with plating efficiencies of 20-50% in vitro and 10-25% when cultured directly from solid tumors. These cells formed adenocarcinomas in vivo. The radiosensitivities in vitro of HLAC-4 and HLAC-14 varied; observed D0 (the inverse of the slope of the exponential portion of the survival curve) values were 80 and 155 rads, respectively; n (the dose at which the exponential portion of the survival curve extrapolates to 100% survival) values were approximately 1.8. Survival curves obtained following in situ irradiation of 4- to 5-mm3 HLAC-4 tumors showed a D0 of 80 rads and an n of 7. Morphology and growth characteristics of two HLAC cell lines in vivo and in vitro were described.

Enhancement of benzo[a]pyrene-induced sister chromatid exchanges in lymphocytes from cigarette smokers occupationally exposed to asbestos.

The frequencies of base-line and benzo[a]pyrene [(BP) CAS 50-38-8]-induced sister chromatid exchanges (SCE) were measured in peripheral blood lymphocytes from 22 male asbestos-exposed workers and 10 nonexposed workers of comparable age. A clear association between cigarette smoking and asbestos exposure in the sensitivity of lymphocytes to BP was observed. Among asbestos-exposed workers, lymphocytes from those who smoked cigarettes were significantly more susceptible to the induction of SCE by in vitro exposure to BP (P = .01) than were lymphocytes from nonsmokers. Active smoking elevated the base-line SCE frequency in both asbestos-exposed and nonexposed workers (P = .001), and an interaction between smoking and asbestos in the production of base-line SCE was suggested (P = .07). Asbestos exposure alone was not associated with an enhanced susceptibility to the induction of SCE by BP or with an elevation of base-line SCE. Increased age was associated with an increase in SCE inducibility by BP (P = .01), and a history of smoking was marginally associated with SCE inducibility by BP (P = .07). These findings support the hypothesis that an increased susceptibility of asbestos-exposed individuals to polyaromatic hydrocarbon-induced cancer results from an enhanced sensitivity to the induction of genetic damage rather than to an asbestos-induced differential cellular metabolic capacity.

Heterogeneous response to X-ray and ultraviolet light irradiations of cultured skin fibroblasts in two families with Gardner's Syndrome.

A heterogeneous response to X-ray and far UV (254 nm) light irradiations was found in cultured skin fibroblast lines from 2 separate families with Gardner's syndrome. When compared to 2 normal control cultures and cultures from 2 patients with nonfamilial colon cancer, cultures from 4 clinically affected members of family 1 showed increased sensitivity to the lethal effects of both X-ray and UV light irradiations. These cells also showed a delayed pattern of X-ray potentially lethal damage repair (PLDR) and absent UV PLDR. In contrast, cultures from 3 members of family 2 (2 of whom were clinically affected) showed a normal response of survival and PLDR to both X-ray and UV light irradiations. Thus increased sensitivity of cultured skin fibroblasts to X-ray and UV light irradiations was not a consistent in vitro finding in patients with Gardner's syndrome. However, in families with Gardner's syndrome who demonstrate in vitro radiosensitivity, additional studies are needed to assess the usefulness of these techniques in detecting affected individuals prior to the development of colon carcinoma and other manifestations.

Quantitative studies of radiation transformation with the A31-11 mouse BALB/3T3 cell line.

A cloned mouse embryo-derived fibroblast cell line was used to study morphological transformation induced by X-rays and 254-nm ultraviolet light (UV). The transformation frequency increased exponentially with increasing dose from 10 to 400 rads for X-rays and 1.0 to 7.5 J/sq m for UV exposure. Split-dose X-ray exposures led to an enhancement in transformation at total doses below 100 rads and a reduction at doses of 300 to 400 rads. The induced transformation frequency varied among serum lots and was very dependent upon the initial cell density. Spontaneous transformants were observed in 10 of 22 consecutive experiments; the spontaneous transformation frequency was generally about 1 to 2 X 10(-5) as compared to induced frequencies which ranged up to 3 X 10(-3) for X-rays and 7.5 X 10(-4) for UV exposure. Further results indicate that this cell line has several potential advantages over the mouse 10T1/2 line for studies with relatively weak in vitro carcinogens such as radiation. These include (a) a reduced overall expression time for the appearance of transformed foci (4 weeks); (b) a high cloning efficiency (50 to 60%); and (c) the fact that about 20 times as many viable cells may be plated per dish for optimal results, allowing transformation frequencies as low as 10(-5) to be measured easily. On the other hand, there was more variability in the results among experiments with the 3T3 cell line.

In vivo enhancement of genomic instability in minisatellite sequences of mouse C3H/10T1/2 cells transformed in vitro by X-rays.

The level of genomic instability was determined during tumor development in vivo. Genomic rearrangements, a marker of genomic instability, was measured in mouse C3H/10T1/2 cells transformed in vitro by X-rays with a DNA fingerprinting assay. Three transformed clones isolated from type III foci were divided into two groups. Cells from the first group were injected s.c. into syngeneic and nonimmunosuppressed C3H mice. After 3 to 5 months, the tumors were excised, and the neoplastic cells were isolated and subcloned. Cells from the second group were incubated in vitro for 25 passages (about 6 months) to approximate the number of cell divisions occurring in the tumor, and then they were subcloned. DNA was extracted from subclones grown in vitro and in vivo and analyzed with the DNA fingerprinting assay. A high frequency of genomic rearrangements (50-100%) was found in subclones derived from tumors that arose in vivo, whereas the frequency was very low (< 10%) among subclones passaged in vitro, suggesting that genetic instability may be enhanced by factors present in the C3H mouse. In one clone (F-17) genomic instability appeared to be activated and down regulated. The high frequency of instability found in tumor cell subclones did not appear to result from an in vivo selection of a more tumorigenic subpopulation of cells present in the original clone prior to injection in the animal. This enhancement of genomic instability occurring in vivo could be required to complete the process of transformation to tumorigenicity and allow the neoplastic cells to adapt to a new environment.

Genomic rearrangements in mouse C3H/10T1/2 cells transformed by X-rays, UV-C, and 3-methylcholanthrene, detected by a DNA fingerprint assay.

Genomic rearrangements occurring in C3H/10T1/2 cells transformed by X-rays were examined with a DNA fingerprint assay. Four multilocus and multiallele probes were employed (M, X, H10, and H16) that detect different families of minisatellite sequences dispersed throughout the genome. Genomic rearrangements were detectable only with probe M. This specificity may be explained by a genomic instability owing to a specific sequence or structure of DNA recognized by probe M. Genomic rearrangements were detected in 5 of 12 type III foci transformed by 600 cGy of X-rays and in all clones isolated from a previously transformed clone exposed to a second dose of 600 cGy and recloned. The latter data suggest that the stage of transformation and the occurrence of genomic rearrangement induced by X-rays may be related. An intensity shift or a complete deletion of band 2 was common to these X-ray-induced clones, as well as to clones transformed by UV-C (1 of 5) or 3-methylcholanthrene (4 of 6). This band did not hybridize to probes for the retinoblastoma gene RB or for p53. We hypothesize that the loss of band 2 may reflect a significant genetic change in the transformation of 10T1/2 cells, perhaps representing the inactivation of a tumor suppressor gene other than RB or p53. Additional rearrangements occurred in X-ray-transformed clones; these rearrangements were not observed with the other carcinogens. Aside from the changes in band 2, however, no specific pattern of genomic rearrangement was associated with X-ray transformation, and the presence or absence of rearrangements did not correlate with tumorigenicity in syngeneic nonimmunosuppressed C3H mice.

Radiosensitivities of ten apparently normal human diploid fibroblast strains to cell killing, G2-phase chromosomal aberrations, and cell cycle delay.

We have examined the sensitivity to X-irradiation of ten apparently normal human fibroblast cell strains received as coded samples. Three different end points were examined: cytotoxicity; induction of chromosomal aberrations after irradiation in G2; and G1-phase block after irradiation and release from confluent growth. Three of these ten strains showed a moderate degree of hypersensitivity to X-rays by all three assays, falling within the range previously reported for ataxia telangiectasia heterozygotes. This variability in the "normal" response may render difficult the use of these techniques in the detection of gene carriers for such disorders in the general population. Furthermore, it indicates that care should be taken in the selection of reference control strains in studies of the sensitivity of cells from various genetic disorders to radiation.

Induction of sister chromatid exchanges by extremely low doses of alpha-particles.

The induction of sister chromatid exchanges (SCE) was examined in Chinese hamster ovary cells irradiated in the G1 phase of the cell cycle with alpha-particles from a plutonium-238 source. A significant increase in the frequency of SCE occurred with doses as low as 0.31 mGy (31 millirads). Although 30% of the cells showed an increased frequency of SCE at this dose, less than 1% of cell nuclei were actually traversed by an alpha-particle. A dose of approximately 2.0 Gy was necessary to produce a similar increase in SCE by X-rays. These results indicate that genetic damage may be induced by low doses of alpha-radiation in cell nuclei not actually traversed by an alpha-particle. This phenomenon may have important implications in the estimation of risks of such exposures.

Characteristics of human diploid fibroblasts transformed in vitro by chemical carcinogens.

We investigated several potential methods to select for carcinogen-induced changes in N-acetoxy-2-acetylaminofluorene-treated normal human diploid fibroblasts, in an effort to isolate cells exhibiting the transformed phenotype. Treated cultures exhibited an extended but not indefinite life span, as well as an increased cloning efficiency in reduced calcium concentrations at 40 to 50 population doublings posttreatment. Morphologically altered foci in monolayer culture, the capacity to proliferate under reduced serum or calcium concentrations, or the ability to grow on irradiated 3T3 monolayers did not uniquely identify or select for a carcinogen-induced phenotype. Treated cultures did routinely produce viable colonies when assayed under anchorage-independent (AI) conditions. This AI phenotype persisted for at least 2 months; when cells from such colonies were isolated and retested, a 2-fold enhancement in the frequency of AI growth was observed. AI-derived cells showed no stable morphological alteration in monolayer culture as regards either growth pattern or cytology. Four out of 10 strains of cells derived from AI colonies and grown to sufficient numbers in monolayer for tumorigenicity testing produced tumors in nude mice; only one of these was invasive and grew progressively to greater than 1 cm in diameter. Cells recovered from these tumors were diploid and of fibroblastic morphology. The AI phenotype appears to be an early marker for a carcinogen-induced change in human fibroblasts, but it is not systematically associated with the other phenotypic characteristics of transformation usually found concomitantly in rodent cell systems.

Characterization of a quantitative assay for the in vitro transformation of normal human diploid fibroblasts to anchorage independence by chemical carcinogens.

We have characterized an assay for the quantitative measurement of the frequency of conversion to anchorage-independent growth of N-acetoxy-2-acetylaminofluorene-treated normal human diploid fibroblasts. We investigated the effects of the following parameters on the absolute number and on the frequency of anchorage-independent colonies scored: (a) the number of cells seeded per dish; (b) the type of posttreatment medium; (c) the number of population doublings allowed posttreatment prior to seeding in suspension; and (d) the carcinogen dose. The assay was linear over the range of 1.9 X 10(3) to 3.8 X 10(4) cells seeded per 6-mm dish for both total colonies scored and the induced frequency of anchorage-independent growth. The medium used posttreatment affected both the frequency and the kinetics of appearance of the anchorage-independent phenotype. The number of population doublings and the number of days allowed posttreatment prior to assaying for anchorage-independent growth potential also influenced the frequency of recovery of this phenotype. Under standardized conditions, the assay yielded a dose-response relationship for transformation to anchorage independence over the concentration range of 0 to 10 microM N-acetoxy-2-acetylaminofluorene.

DNA-protein cross-linking by chemical carcinogens in mammalian cells.

The induction of DNA cross-linking in mammalian cells by various carcinogens was investigated by the method of alkaline elution. A dose-dependent increase in DNA cross-linking was seen following exposure of human fibroblasts to N-acetyoxy-2-acetylaminofluorene and following exposure of mouse embryo cells to 7,12-dimethylbenz[a]-anthracene. No cross-link effect was seen following treatment with N-methyl-N'-nitro-N-nitrosoguanidine, benz-[a]anthracene, benz[A]anthracene-5,6-dihydroepoxide, or metabolic inhibitors. The cross-linking appeared to be DNA-protein in nature since proteinase treatment removed the effect. DNA single-strand breaks were also induced by several of these agents in the case of N-acetoxy-2-acetylaminofluorene and N-methyl-N'-nitro-N-nitrosoguanidine, approximately 70 to 90% of these breaks were rejoined after an 18-hr incubation in fresh medium, whereas repair of the cross-links induced by N-acetoxy-2-acetylaminofluorene was slight at this time.

Enhancement of X-ray-induced transformation in C3H/10T1/2 cells by interferon.

Interferon may enhance malignant transformation induced by X-rays in a C3H mouse embryo-derived cell line. The inhibitory effect of interferon on cell division during the proliferative phase of the expression of the transformational damage may be of importance in the understanding of this finding.

Localization of polycyclic hydrocarbon carcinogens in the lung following intratracheal instillation in gelatin solution.

The deposition and localization of benzo(a)pyrene (BP) suspended in a gelatin-0.9 percent NaCl solution was studied in hamster lungs by ultraviolet fluorescence microscopy. BP was deposited primarily in the alveolar region of the lung. Although large numbers of BP-filled macrophages were seen in the upper airways by 24 hr after an instillation, little BP appeared to penetrate into the bronchial epithelium. The intratracheal instillation of polycyclic hydrocarbons in a gelatin-0.9 percent NaCl solution appears to be a useful model when it is desired to deliver the carcinogen dose to the peripheral lung.