J. Leslie Redpath

Low-Dose Reduction in Transformation Frequency Compared to Unirradiated Controls: The Role of Hyper-radiosensitivity to Cell Death

Abstract Redpath, J. L., Short, S. C., Woodcock, M. and Johnston, P. J. Low-Dose Reduction in Transformation Frequency Compared to Unirradiated Controls: The Role of Hyper-radiosensitivity to Cell Death. Radiat. Res. 159, 433–436 (2003). Calculations based on plausible parameters taken from the existing experimental database, and new measurements on the cell cycle dependence of low-dose hyper-radiosensitivity (HRS) of non-tumorigenic HeLa × skin fibroblast human hybrid cells, provide the first experimental evidence that the selective killing of a transformation-sensitive G2/M-phase subpopulation as a consequence of low-dose HRS could account in part for the observed reduction of induced transformation frequencies at low doses to values below that observed spontaneously. However, it is clear that other mechanisms associated with classical adaptive response, such as induced DNA repair, are also likely to be involved.

ATR dependent activation of Chk2.

ATM and ATR are essential regulators of DNA damage checkpoints in mammalian cells through their respective effectors, Chk2 and Chk1. Cross regulation of the ATM‐Chk2 and ATR‐Chk1 pathways is very limited, although ATM and ATR show overlapping function in a partnership and time‐dependent manner. In this study, we report that Chk2 is a substrate of ATR in response to ionizing and ultraviolet radiation. ATR activation induced by ionizing radiation (IR) is weak in ATM+/+ cells. However, when ATM is inhibited by caffeine, ATR activation is markedly enhanced. Total Chk2 and Chk2 Thr68 are also hyperphosphorylated in the presence of caffeine. Both ATM+/+ and ATM−/− cells display normal ATR activation in response to UV radiation‐induced DNA damage, which is caffeine sensitive. In two lines of ATM‐deficient, as well as in an ATM siRNA silencing cell line, ATR is activated when the cells are exposed to IR and is able to phosphorylate Chk2 in vitro. These observations suggest that ATR is one of the kinases that is likely involved in phosphorylation of Chk2 in response to IR when ATM is deficient. J. Cell. Physiol. 208: 613–619, 2006.

A simplified and rapid staining method for the HeLa × skin fibroblast human hybrid cell neoplastic transformation assay

A simplified and rapid screening method for detecting radiation-induced neoplastically transformed foci in the HeLa x skin fibroblast human hybrid cell assay system has been developed. The method is based on the recent identification of the tumor-associated antigen in this system as intestinal alkaline phosphatase (IAP), and on the recent commercial development of a stable alkaline phosphatase chromogenic substrate solution, Western blue (WB). Cleavage of the substrate results in the production of a blue insoluble precipitate. It is shown that WB can be used on both viable and paraformaldehyde-fixed cells. Fixation does not noticeably reduce the IAP enzymatic activity. A direct comparison with the current method of immunoperoxidase (IMPO) staining indicates that the WB method is not only easier, but appears to be more sensitive in picking up weakly positive foci with a resulting higher (factor of 2.5) induced transformation frequency for 7 Gy of 137Cs gamma radiation. Whereas the IMPO staining procedure is time-consuming and requires access to large amounts of expensive IAP-specific BD6 monoclonal antibody and peroxidase-labeled secondary antibody, the WB staining procedure is rapid and utilizes an inexpensive and readily available reagent. It should now allow this assay system to enter general use.

The effect of adriamycin on radiation damage to mouse lung and skin

Abstract Adriamycin (doxorubicin HCl) (10 mg/kg) administered i.p. to C 3 H HeJ mice 24 hr before or after thoracic radiation has been found to reduce the LD 50 40−180 i.e. the dose to kill 50% animals through pulmonary damage, from 1600 rad to 960 rad. A similar dose of Adriamycin administered i.p. 0.5–1 hour before irradiation was found not to enhance the skin reaction of male ICR albino mice feet.

Subadditive interaction of radiation and taxol in vitro

PURPOSE To examine the dependency of Taxol-radiation interactions on the scheduling of the two agents. METHODS AND MATERIALS The human laryngeal squamous cell carcinoma line SCC20 was used for this study. Cells were irradiated as subconfluent cultures using Cs-137 gamma rays at a dose rate of 1.75 Gy/min. Cultures were pretreated with Taxol (7.5 nM for 12 h, S.F. = 0.4) and then irradiated with graded doses followed by either immediate plating or holding for 6 h either in the absence or presence of 7.5 nM Taxol prior to plating for colony-forming ability. Experiments in which cells were irradiated and then exposed to 7.5 nM Taxol for both 12 and 18 h were also performed. Parallel-flow cytometric analyses of cell-cycle distribution of the various treated populations were carried out. RESULTS The results indicate that pretreatment with Taxol induced a G2 block which was maintained during 6 h postirradiation holding either in the presence or absence of Taxol. No modification of radiosensitivity in the low-dose region was seen for cells treated with Taxol, irradiated, and plated immediately, with the resulting survival being compatible with an additive effect. However, for Taxol-pretreated cells held for 6 h postirradiation, either in the absence or presence of Taxol, the resulting survival reproducibly demonstrated a marked less than additive effect. This was particularly prominent for cells held in the presence of Taxol. Subsequent experiments in which Taxol was added to cells immediately postirradiation again demonstrated a less than additive effect of the two modalities. CONCLUSION The results of this study are consistent with a dual mechanism of action involving Taxol-induced radiation resistance, possibly as a consequence of postirradiation holding in G2, and radiation-induced Taxol resistance through an as-yet-undefined mechanism.

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.

Loss of suppressor loci on chromosomes 11 and 14 may be required for radiation-induced neoplastic transformation of HeLa x skin fibroblast human cell hybrids

We have previously reported a linkage between radiation-induced damage to a putative tumor suppressor locus on fibroblast chromosome 11 and the re-expression of tumorigenicity in a hybrid cell line (HeLa x human skin fibroblast) used to study neoplastic transformation. Further investigation into the molecular basis of radiation-induced neoplastic transformation of the hybrid cell, CGL1, indicates that loss of fibroblast chromosome 11 appears to be necessary but not sufficient for neoplastic transformation. Previous analysis had suggested, though not clearly demonstrated, a possible role for loss of alleles on fibroblast chromosome 14 in the neoplastic transformation of the hybrid cells. Therefore, the status of chromosome 14 in the gamma-ray-induced, neoplastically transformed (GIM) hybrid cell lines and in nontumorigenic control (CON) hybrid cell lines isolated from irradiated populations has been investigated. Chromosome painting and molecular studies using restriction fragment length polymorphisms and tetranucleotide repeat polymorphism analysis were performed. As an additional control, the status of chromosome 12 was also examined. We report that five of the eight GIM cell lines have lost one complete copy of a fibroblast chromosome 14 while only one of the five CON cell lines has lost a complete copy of a fibroblast chromosome 14. No evidence of large-scale loss of chromosome 12 was detected in the GIM or CON cells. The data further suggest that both copies of fibroblast chromosome 14 contain an active tumor suppressor locus and that radiation-induced loss of either fibroblast chromosome 14 is associated with neoplastic transformation in this system. We now conclude that loss of alleles on both fibroblast chromosome 11 and 14 may be required for the radiation-induced neoplastic transformation of these human hybrid cells.

Differential gene expression in tumorigenic and nontumorigenic HeLa × normal human fibroblast hybrid cells

Fusion of tumorigenic HeLa cells with human skin fibroblasts results in chromosomally stable hybrids that are nontumorigenic and no longer express the HeLa tumor‐associated marker intestinal alkaline phosphatase (IAP). Previous studies of spontaneous tumorigenic segregants from the nontumorigenic hybrids implicated the loss of one copy of human fibroblast chromosome 11 in the concomitant reexpression of tumorigenicity. In an attempt to identify genes involved in the control of tumorigenic expression, we performed differential display screening of nontumorigenic hybrid cells and tumorigenic segregants. Subsequent northern blot analyses reproducibly showed 17 differentially expressed genes, eight of which were expressed differentially in the nontumorigenic hybrids and nine of which were expressed differentially in the tumorigenic hybrids. The former were genes for 80K‐L protein (a substrate of protein kinase C), AXL/UFO (a receptor tyrosine kinase), insulin‐like growth factor binding protein 3, apolipoprotein AI regulatory protein, collagen type I α‐2 chain, transforming growth factor‐β–induced gene product 3 (BIGH3), pregnancy‐specific β‐1‐glycoprotein, and fibroblast activation protein α. The latter nine genes were genes for serum/glucocorticoid‐regulated kinase (SGK; a serine/threonine protein kinase), PTPCAAX1 (a tyrosine phosphatase), CXCR‐4 (a G‐protein–coupled membrane receptor), L‐kynurenine hydrolase, β‐1,4‐galactosyltransferase, keratin 8, keratin 17, and H19 and a novel gene. The differential expression of these genes provided several interesting candidates for regulation of tumorigenic expression, including those involved in signal transduction and the extracellular matrix, cytoskeletal proteins, cell‐surface enzyme, and the H19 gene. Mol. Carcinog. 26:298–304, 1999.

Inactivation of the Cystatin E/M Tumor Suppressor Gene in Cervical Cancer

We have previously localized a cervical cancer tumor suppressor gene to a 300 kb interval of 11q13. Analysis of candidate genes revealed loss of expression of cystatin E/M, a lysosomal cysteine protease inhibitor, in 6 cervical cancer cell lines and 9 of 11 primary cervical tumors. Examination of the three exons in four cervical cancer cell lines, 19 primary tumors, and 21 normal controls revealed homozygous deletion of exon 1 sequences in one tumor. Point mutations were observed in six other tumors. Two tumors contained mutations at the consensus binding sites for cathepsin L, a lysosomal protease overexpressed in cervical cancer. Introduction of these two point mutations using site directed mutagenesis resulted in reduced binding of mutated cystatin E/M to cathepsin L. Although mutations were not observed in any cell lines, four cell lines and 12 of 18 tumors contained promoter hypermethylation. Reexpression of cystatin E/M was observed after 5′aza 2‐deoxycytidiene and/or Trichostatin A treatment of cervical cancer cell lines, HeLa and SiHa, confirming promoter hypermethylation. Ectopic expression of cystatin E/M in these two cell lines resulted in growth suppression. There was also suppression of soft agar colony formation by HeLa cells expressing the cystatin E/M gene. Reexpression of cystatin E/M resulted in decreased intracellular and extracellular expression of cathepsin L. Overexpression of cathepsin L resulted in increased cell growth which was inhibited by the reintroduction of cystatin E/M. We conclude, therefore, that cystatin E/M is a cervical cancer suppressor gene and that the gene is inactivated by somatic mutations and promoter hypermethylation.

The effect of adriamycin and actinomycin D on radiation-induced skin reactions in mouse feet

Abstract The effects of adriamycin (10 mg/kg, i.p.) and actinomycin D (0.7 mg/kg, i.p.) on radiation-induced skin reactions in mouse feet have been examined. Drugs were administered either 24 or 2 hr before, or 24 hr after single doses of 6 MeV photons. Split radiation dose experiments were also performed with the drug being administered either 24 or 2 hr before the first radiation dose. A five daily fraction experiment with drug administered 2 hr before the first and third doses was also carried out. In none of these experiments did adriamycin alter the kinetics of appearance, or the severity of, the radiation-induced reaction. Furthermore, drug administered 30 days after the radiation dose, i.e. after the radiation reaction was healed, did not induce any further reaction. These results confirm our previous preliminary observations that adriamycin does not significantly enhance radiation damage to the skin of mouse feet. In the case of actinomycin D no enhancement of skin reaction in single radiation dose experiments was observed. However, in both the split dose and five fraction study a significantly more severe skin reaction was seen in the drug treated animals compared to those treated with radiation alone.