Jeffrey L. Schwartz

Selenium and GPx-1 overexpression protect mammalian cells against UV-induced DNA damage

Supplementation of the culture media of human MCF-7 breast carcinoma cells or mouse fibroblasts with low levels of selenium (30 nM) provided as sodium selenite was shown to protect these cells from ultraviolet (UV)-induced chromosome damage, as quantified by micronucleus assay. Selenium supplementation was also effective in reducing UV-induced gene mutations as measured in the lacI shuttle vector model. Protection was dependent on functional BRCA1 activity, a protein implicated in breast cancer risk and DNA damage repair. In addition, overexpression of GPx-1, a selenoprotein with antioxidant activity, also attenuated UVinduced micronuclei formation in the absence of selenium supplementation. Combining selenium supplementation with GPx-1 overexpression further reduced UV-induced micronucleus frequency. These data provide evidence that the benefits of selenium supplementation might be either through the prevention or repair of DNA damage, and they implicate at least one selenoprotein (GPx-1) in the process.

Effects of HOXB4 overexpression on ex vivo expansion and immortalization of hematopoietic cells from different species

Overexpression of the human HOXB4 has been shown to induce the expansion and self‐renewal of murine hematopoietic stem cells. In preparation for clinical studies, we wished to investigate the effects of HOXB4 on cells from other species, in particular preclinical large animals such as dogs and nonhuman primates. Thus, we transduced CD34+ cells from nonhuman primates, dogs, and humans with a HOXB4‐expressing gammaretroviral vector and a yellow fluorescent protein‐expressing control vector. Compared with the control vector, HOXB4 overexpression resulted in a much larger increase in colony‐forming cells in dog cells (28‐fold) compared with human peripheral blood, human cord blood, and baboon cells (two‐, four‐, and fivefold, respectively). Furthermore, we found that HOXB4 overexpression resulted in immortalization with sustained growth (>12 months) of primitive hematopoietic cells from mice and dogs but not from monkeys and humans. This difference correlated with increased levels of retrovirally overexpressed HOXB4 in dog and mouse cells compared with human and nonhuman primate cells. The immortalized cells did not show any evidence of insertional mutagenesis or chromosomal abnormalities. Competitive congenic transplantation experiments showed that HOXB4‐expanded mouse cells engrafted well after 1 or 3 months of expansion, and no leukemia was observed in mice. Our findings suggest that the growth promoting effects of HOXB4 are critically dependent on HOXB4 expression levels and that this can result in important species‐specific differences in potency.

A Role for Endogenous and Radiation-Induced DNA Double-Strand Breaks in p53-Dependent Apoptosis during Cortical Neurogenesis

Abstract Li, H., Liu, N., Rajendran, G. K., Gernon, T. J., Rockhill, J. K., Schwartz, J. L. and Gu, Y. A Role for Endogenous and Radiation-Induced DNA Double-Strand Breaks in p53-Dependent Apoptosis during Cortical Neurogenesis. Radiat. Res. 169, 513–522 (2008). Prenatal exposure to low-dose radiation increases the risk of microcephaly and/or mental retardation. Microcephaly is also associated with genetic mutations that affect the non-homologous end-joining pathway of DNA double-strand break repair. To examine the link between these two causal factors, we characterized the neural developmental effects of acute radiation exposure in mouse littermate embryos harboring mutations in the Ku70 and p53 genes. Both low-dose radiation exposure and Ku70 deficiency induced morphologically indistinguishable cortical neuronal apoptosis. Irradiated Ku70-deficient embryos displayed anatomical damage indicative of increased radiosensitivity in the developing cerebral cortex. Deleting the p53 gene not only rescued cortical neuronal apoptosis at all levels but also restored the in vitro growth of Ku70-deficient embryonic fibroblasts despite the presence of unrepaired DNA/chromosomal breaks. The results confirm the role of DNA double-strand breaks as a common causative agent of apoptosis in the developing cerebral cortex. Furthermore, the findings suggest a disease mechanism by which the presence of endogenous DNA double-strand breaks in the newly generated cortical neurons becomes radiomimetic when DNA end joining is defective. This in turn activates p53-dependent neuronal apoptosis and leads to microcephaly and mental retardation.

Induction and loss of a TP53-dependent radioadaptive response in the human lymphoblastoid cell model TK6 and its abrogation by BCL2 over-expression.

Purpose: To characterize the radioadaptive response in the human lymphoblastoid cell model TK6, and determine: (i) Whether repeated low dose exposures are more effective than single acute exposures in inducing resistance, (ii) the time-course for induction and loss of resistance following chronic exposures, and (iii) the effect of TP53 deletion or BCL2 over-expression on the induction of an adaptive response. Materials and methods: TK6, a human B-lymphoblastoid cell line, TK6-BCL2, a TK6 line that over-expresses BCL2 and is resistant to radiation-induced apoptosis, and NH32, a TP53 knockout of TK6 that is also resistant to apoptosis were studied. Cells were exposed to chronic, daily doses of 10 cGy given over 1 – 21 days before being challenged with 1 – 5 Gy exposures. Cell survival and chromatid break induction following high dose challenge were used to evaluate adaptive radiation responses. Results: Exposure to 10 cGy gamma rays induced resistance to killing and chromosome break induction in TK6 cells, but not in either TK6-BCL2 or NH32 cells. Resistance in TK6 was observed 4 h after exposure, and cells remained resistant for about 48 h. Maximal resistance was induced by a single 10 cGy dose. Repeated 10 cGy exposures had no additional effect on radiation sensitivity, except to maintain the induced radioresistance. Conclusion: An adaptive response is maximally and rapidly induced by a single low dose exposure in TK6 cells, and it has a limited lifespan. Induction of an adaptive response in TK6 cells can be abrogated by either TP53 loss or BCL2 over-expression. The characteristics of induced resistance in TK6 cells suggest that alterations in TP53-dependent apoptotic responses may be one mechanism for resistance.

Atm Haploinsufficiency does not Affect Ionizing Radiation Mutagenesis in Solid Mouse Tissues

Abstract Connolly, L., Lasarev, M., Jordan, R., Schwartz, J. L. and Turker, M. S. Atm Haploinsufficiency does not Affect Ionizing Radiation Mutagenesis in Solid Mouse Tissues. Radiat. Res. 166, 39–46 (2006). Ataxia telangiectasia (AT) is a hereditary disease with autosomal recessive inheritance of ATM (ataxia telangiectasia mutation) alleles. AT is associated with severe sensitivity to ionizing radiation and a strong predisposition to develop cancer. A modest increase in cancer, particularly for the breast, has been shown for ATM carriers (i.e. heterozygotes), and a modest increase in radiation sensitivity has also been shown for those patients and their cells. However, the extent of these effects is unclear. Based on the well-established relationship between cancer and mutation, we used a mouse model for Atm haploinsufficiency to ask whether partial loss of Atm function could lead to an increased mutagenic response for solid tissues of mice exposed to radiation. The autosomal mouse Aprt gene was used as the mutational target and kidney and ear as the target tissues in B6D2F1 hybrids. Although induction of autosomal mutations was readily demonstrated in both tissues, a comparison of these data with those from an identical study performed with B6D2F1 mice that were wild-type for Atm (Cancer Res. 62, 1518–1523, 2002) revealed that Atm haploinsufficiency did not alter the radiation mutagenic response for the cells of either tissue. Moreover, no effect of Atm haploinsufficiency on reduced cellular viability due to radiation exposure was observed. The results demonstrate that Atm haploinsufficiency does not alter the radiation mutagenic response or decrease viability for normally quiescent cells in solid tissues of the mouse.

Tp53 codon-72 polymorphisms identify different radiation sensitivities to g2-chromosome breakage in human lymphoblast cells

Both the G2 chromosomal radiosensitivity assay and allelic differences in TP53 codon‐72 have been associated with cancer predisposition. The relationship between the two endpoints was determined in 56 human EBV‐transformed lymphoblastoid cell lines. Although there were overlapping distributions of sensitivity for the different genotypes, cell lines that were homozygous for the proline coding allele were more likely to be resistant to chromatid break formation than those containing two arginine coding alleles, whereas cell lines expressing both the proline and arginine codon were either resistant like proline–proline lines or sensitive like arginine–arginine lines. The results support an important role of the TP53 codon‐72 polymorphism in modifying G2‐chromosome radiosensitivity. Distinguishing the effect of TP53 codon‐72 variations from other modifiers of G2‐chromosome radiosensitivity might aid in identifying new markers of cancer risk. Environ. Mol. Mutagen., 2010.

Environmental mutagenesis and radiation biology: The legacy of William Morgan

A symposium entitled Environmental Mutagenesis and Radiation Biology was held on September 27, 2016 to honor the memory of Dr. William F. Morgan who passed away unexpectedly on November 13, 2015. The speakers presented the latest reviews on homologous recombination repair, induced genetic instability, bystander effects, and risk estimate development. Their presentations are presented following the introduction.