Eugene Elmore

Low doses of diagnostic energy X-rays protect against neoplastic transformation in vitro.

Purpose : To investigate the effect of low doses of 60 kVp X-rays on in vitro transformation frequency. Materials and methods : HeLa ×skin fibroblast human hybrid cells were used to assay transformation from the non-tumorigenic to the tumorigenic phenotype. Subconfluent cultures of cells were exposed to a range of doses of 60 kVp X-rays and seeded for assay of transformation after 24 h post-irradiation holding. Experiments were repeated at least three times and the data pooled for analysis. Transformation frequencies were compared with those of sham-irradiated controls. Results : At doses < 1 cGy, the observed transformation frequencies were significantly less than those seen in unirradiated cells. Conclusion : Low doses (< 1 cGy) of 60 kVp X-rays protect HeLa ×skin fibroblast human hybrid cells against neoplastic transformation in vitro.

Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues

Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues.

The effect of dose rate on radiation-induced neoplastic transformation in vitro by low doses of low-LET radiation.

Abstract Elmore, E., Lao, X-Y., Kapadia, R. and Redpath, J. L. The Effect of Dose Rate on Radiation-Induced Neoplastic Transformation In Vitro by Low Doses of Low-LET Radiation. Radiat. Res. 166, 832–838 (2006). The dependence of the incidence of radiation-induced cancer on the dose rate of the radiation exposure is a question of considerable importance to the estimation of risk of cancer induction by low-dose-rate radiation. Currently a dose and dose-rate effectiveness factor (DDREF) is used to convert high-dose-rate risk estimates to low dose rates. In this study, the end point of neoplastic transformation in vitro has been used to explore this question. It has been shown previously that for low doses of low-LET radiation delivered at high dose rates, there is a suppression of neoplastic transformation frequency at doses less than around 100 mGy. In the present study, dose–response curves up to a total dose of 1000 mGy have been generated for photons from 125I decay (approximately 30 keV) delivered at doses rates of 0.19, 0.47, 0.91 and 1.9 mGy/min. The results indicate that at dose rates of 1.9 and 0.91 mGy/min the slope of the induction curve is about 1.5 times less than that measured at high dose rate in previous studies with a similar quality of radiation (28 kVp mammographic energy X rays). In the dose region of 0 to 100 mGy, the data were equally well fitted by a threshold or linear no-threshold model. At dose rates of 0.19 and 0.47 mGy/min there was no induction of transformation even at doses up to 1000 mGy, and there was evidence for a possible suppressive effect. These results show that for this in vitro end point the DDREF is very dependent on dose rate and at very low doses and dose rates approaches infinity. The relative risks for the in vitro data compare well with those from epidemiological studies of breast cancer induction by low- and high-dose-rate radiation.

Low doses of very low-dose-rate low-LET radiation suppress radiation-induced neoplastic transformation in vitro and induce an adaptive response

Abstract Elmore, E., Lao, X-Y., Kapadia, R., Giedzinski, E., Limoli, C. and Redpath, J. L. Low Doses of Very Low-Dose-Rate Low-LET Radiation Suppress Radiation-Induced Neoplastic Transformation In Vitro and Induce an Adaptive Response. Radiat. Res. 169, 311–318 (2008). The purpose of this study was to determine whether adaptation against neoplastic transformation could be induced by exposure to very low-dose-rate low-LET radiation. HeLa × skin fibroblast human hybrid cells were irradiated with ∼30 kVp photons from an array of 125I seeds. The initial dose rate was 4 mGy/day. Cell samples were taken at four intervals at various times over a period of 88 days and assayed for neoplastic transformation and the presence of reactive oxygen species (ROS). The dose rate at the end of this treatment period was 1.4 mGy/day. Transformation frequencies and ROS levels were compared to those of parallel unirradiated controls. At the end of 3 months and an accumulated dose of 216 mGy, cells treated with very low-dose-rate radiation were exposed to a high-dose-rate 3-Gy challenge dose of 137Cs γ rays, and the effects compared with the effect of 3 Gy on a parallel culture of previously unirradiated cells. Cells exposed to very low-dose-rate radiation exhibited a trend toward a reduction in neoplastic transformation frequency compared to the unirradiated controls. This reduction seemed to diminish with time, indicating that the dose rate, rather than accumulated dose, may be the more important factor in eliciting an adaptive response. This pattern was in general paralleled by a reduction of ROS present in the irradiated cultures compared to controls. The very low-dose-rate-treated cells were less sensitive to the high challenge dose than unirradiated controls, suggesting the induction of an adaptive response. Since there was a suggestion of a dose-rate threshold for induction suppression, a second experiment was run with a fresh batch of cells at an initial dose rate of 1 mGy/day. These cells were allowed to accumulate 40 mGy over 46 days (average dose rate = 0.87 mGy/day), and there was no evidence for suppression of transformation frequency compared to parallel unirradiated controls. It is concluded that doses of less than 100 mGy delivered at very low dose rates in the range 1 to 4 mGy/day can induce an adaptive response against neoplastic transformation in vitro. When the dose rate drops below ∼1 mGy/day, this suppression is apparently lost, suggesting a possible dose-rate-dependent threshold for this process.

Match criteria for human cell line authentication: Where do we draw the line?†‡

Continuous human cell lines have been used extensively as models for biomedical research. In working with these cell lines, researchers are often unaware of the risk of cross‐contamination and other causes of misidentification. To reduce this risk, there is a pressing need to authenticate cell lines, comparing the sample handled in the laboratory to a previously tested sample. The American Type Culture Collection Standards Development Organization Workgroup ASN‐0002 has developed a Standard for human cell line authentication, recommending short tandem repeat (STR) profiling for authentication of human cell lines. However, there are known limitations to the technique when applied to cultured samples, including possible genetic drift with passage. In our study, a dataset of 2,279 STR profiles from four cell banks was used to assess the effectiveness of the match criteria recommended within the Standard. Of these 2,279 STR profiles, 1,157 were grouped into sets of related cell lines—duplicate holdings, legitimately related samples or misidentified cell lines. Eight core STR loci plus amelogenin were used to unequivocally authenticate 98% of these related sets. Two simple match algorithms each clearly discriminated between related and unrelated samples, with separation between related samples at ≥80% match and unrelated samples at <50% match. A small degree of overlap was noted at 50–79% match, mostly from cell lines known to display variable STR profiles. These match criteria are recommended as a simple and effective way to interpret results from STR profiling of human cell lines.

Neoplastic Transformation In Vitro after Exposure to Low Doses of Mammographic-Energy X Rays: Quantitative and Mechanistic Aspects

Abstract Ko, S. J., Liao, X-Y., Molloi, S., Elmore, E. and Redpath, J. L. Neoplastic Transformation In Vitro after Exposure to Low Doses of Mammographic-Energy X Rays: Quantitative and Mechanistic Aspects. Radiat. Res. 162, 646–654 (2004). The induction of neoplastic transformation in vitro after exposure of HeLa × skin fibroblast hybrid cells to low doses of mammography-energy (28 kVp) X rays has been studied. The data indicate no evidence of an increase in transformation frequency over the range 0.05 to 22 cGy, and doses in the range 0.05 to 1.1 cGy may result in suppression of transformation frequencies to levels below that seen spontaneously. This finding is not consistent with a linear, no-threshold dose– response curve. The dose range at which possible suppression is evident includes doses typically experienced in mammographic examination of the human breast. Experiments are described that attempt to elucidate any possible role of bystander effects in modulating this low-dose radiation response. Not unexpectedly, inhibition of gap junction intercellular communication (GJIC) with the inhibitor lindane did not result in any significant alteration of transformation frequencies seen at doses of 0.27 or 5.4 cGy in these subconfluent cultures. Furthermore, no evidence of a bystander effect associated with factors secreted into the extracellular medium was seen in medium transfer experiments. Thus, in this system and under the experimental conditions used, bystander effects would not appear to be playing a major role in modulating the shape of the dose–response curve.

Neoplastic transformation in vitro induced by low doses of 232 MeV protons

The aim was to define the dose – response curve for high-energy proton-induced neoplastic transformation in vitro. The HeLa × skin fibroblast human hybrid cell assay was used to determine the frequency of neoplastic transformation following doses of 232 MeV protons (mean linear energy transfer, LET = 0.44 keV μm – 1) in the range 5 – 600 mGy. Proton irradiations were carried out at the Loma Linda University Proton Treatment Facility, CA, USA. The data indicate no evidence for induction of transformation below a dose of 100 mGy. At doses of 5 and 50 mGy, there is evidence for a possible suppression of transformation frequencies below that for spontaneous transformation. The shape of the dose – response curve for high-energy proton-induced transformation of the human hybrid cell line CGL1 does not follow a linear no-threshold model and shows evidence for a threshold as well as for possible suppression of transformation at doses < 100 mGy, similar to that seen for other low-LET radiations.

RADIATION-INDUCED NEOPLASTIC TRANSFORMATION IN VITRO, HORMESIS AND RISK ASSESSMENT

Dose-response curves for various low-LET radiation sources have consistently been demonstrated to be J-shaped for the cancer-relevant endpoint of neoplastic transformation in vitro. Most of these studies have been performed where the radiation has been delivered at intermediate to high dose-rates (30–3000 mGy/min), where the threshold dose for induction of neoplastic transformation is around 100–200 mGy. Below these doses, the transformation frequency is less than that seen spontaneously, indicative of a hormetic effect. More recently, data have been obtained for low dose rates (<0.5 mGy/min) of low-LET radiation, and again hormetic effects are apparent but with threshold doses now being >1000 mGy. Similar trends have been reported in animal experiments as well as in human epidemiologic studies. Indeed, the relative risks for induction of neoplastic transformation in vitro in the dose range 1 to 1000 mGy agree well with those for incidence of radiation-induced breast cancer and leukemia in humans. These findings support the notion that the endpoint of neoplastic transformation in vitro is a plausible endpoint to not only study mechanisms involved in response to low doses of radiation, but also to provide information of potential importance to risk assessment.

Threshold-Type Dose Response for Induction of Neoplastic Transformation by 1 GeV/nucleon Iron Ions

Abstract Elmore, E., Lao, X-Y., Kapadia, R. and Redpath, J. L. Threshold-Type Dose Response for Induction of Neoplastic Transformation by 1 GeV/nucleon Iron Ions. Radiat. Res. 171, 764ndash;770 (2009). Neoplastic transformation of HeLa × skin fibroblast human hybrid cells by doses of 1 GeV/nucleon iron ions in the range 1 cGy to 1 Gy to exposed cultures has been examined. The data indicate a threshold-type dose–response curve with no increase in transformation frequency until doses above 20 cGy. At doses <10 cGy, not all exposed cells receive a direct traversal of an iron-ion track core, but all exposed cells receive up to several mGy of low-LET radiation associated with the δ-ray penumbra. It is proposed that the threshold-type response seen is a consequence of an adaptive response associated with the δ-ray exposure. For comparison purposes, the dose response for 137Cs γ rays over the same dose range was examined using the same experimental procedure. As we have shown previously, the dose response for 137Cs γ radiation was J-shaped. The iron ions were 1.5 to 1.7 times more biologically effective than the γ radiation over the dose range examined.

The human epithelial cell cytotoxicity assay for determining tissue specific toxicity

The Human Epithelial Cell Cytotoxicity (HECC) Assay for determining organ specific cytotoxicity uses human epithelial cells from eight different human tissues, including: skin, mammary, prostate, renal, bronchial, oral, ecto-cervix, and liver. Although the initial studies using this assay were conducted using cancer chemopreventive agents, the HECC Assay can also be used to evaluate other types of drugs, personal care products, environmental chemicals, and potential toxicants. Human epithelial cells at an early passage are seeded into multi-well dishes. The cells are exposed to multiple concentrations of a test agent for a three day period. The concentration ranges for test agents in the assay are determined in a preliminary assay using an exposure of five days and log dilutions from the highest soluble concentration. At the end of the exposure period, the cultures are evaluated for inhibition of growth. In the HECC Assay, cultures are exposed for three days. At the end of the exposure period, the cultures are evaluated for inhibition of growth, mitochondrial function, and PCNA expression or albumin synthesis (hepatocytes). Data are analyzed to determine the concentration that inhibited and point by 50 percent (TC(50)). Values for each agent in each target epithelial cell line or culture and the target tissue specific sensitivity are compared to determine the relative sensitivity of each epithelial cell line to the test agent.