Nigel E. A. Crompton

CD4 and CD8 T-lymphocyte apoptosis can predict radiation-induced late toxicity: a prospective study in 399 patients.

Purpose: Predicting late effects in patients treated with radiation therapy by assessing in vitro radiation-induced CD4 and CD8 T-lymphocyte apoptosis can be useful in individualizing treatment. Experimental Design: In a prospective study, 399 curatively irradiated patients were tested using a rapid assay where fresh blood samples were in vitro irradiated with 8 Gy X-rays. Lymphocytes were collected and prepared for flow cytometric analysis. Apoptosis was assessed by associated condensation of DNA. The incidences of late toxicities were compared for CD4 and CD8 T-lymphocyte apoptoses using receiver-operating characteristic curves and cumulative incidence. Results: No association was found between early toxicity and T-lymphocyte apoptosis. Grade 2 and 3 late toxicities were observed in 31% and 7% of patients, respectively. More radiation-induced T-lymphocyte apoptosis was significantly associated with less grade 2 and 3 late toxicity (Grays test, P < 0.0001). CD8 (area under the curve = 0.83) was more sensitive and specific than CD4. No grade 3 late toxicity was observed for patients with CD4 and CD8 values greater than 15% and 24%, respectively. The 2-year cumulative incidence for grade 2 or 3 late toxicity was 70%, 32%, and 12% for patients with absolute change in CD8 T-lymphocyte apoptosis of ≤16, 16 to 24, and >24, respectively. Conclusions: Radiation-induced T-lymphocyte apoptosis can significantly predict differences in late toxicity between individuals. It could be used as a rapid screen for hypersensitive patients to radiotherapy. In future dose escalation studies, patients could be selected using the apoptosis assay.

Single nucleotide polymorphisms, apoptosis, and the development of severe late adverse effects after radiotherapy.

Purpose: Evidence has accumulated in recent years suggestive of a genetic basis for a susceptibility to the development of radiation injury after cancer radiotherapy. The purpose of this study was to assess whether patients with severe radiation-induced sequelae (RIS; i.e., National Cancer Institute/CTCv3.0 grade, ≥3) display both a low capacity of radiation-induced CD8 lymphocyte apoptosis (RILA) in vitro and possess certain single nucleotide polymorphisms (SNP) located in candidate genes associated with the response of cells to radiation. Experimental Design: DNA was isolated from blood samples obtained from patients (n = 399) included in the Swiss prospective study evaluating the predictive effect of in vitro RILA and RIS. SNPs in the ATM, SOD2, XRCC1, XRCC3, TGFB1, and RAD21 genes were screened in patients who experienced severe RIS (group A, n = 16) and control subjects who did not manifest any evidence of RIS (group B, n = 18). Results: Overall, 13 and 21 patients were found to possess a total of <4 and ≥4 SNPs in the candidate genes. The median (range) RILA in group A was 9.4% (5.3-16.5) and 94% (95% confidence interval, 70-100) of the patients (15 of 16) had ≥4 SNPs. In group B, median (range) RILA was 25.7% (20.2-43.2) and 33% (95% confidence interval, 13-59) of patients (6 of 18) had ≥4 SNPs (P < 0.001). Conclusions: The results of this study suggest that patients with severe RIS possess 4 or more SNPs in candidate genes and low radiation-induced CD8 lymphocyte apoptosis in vitro.

Altered apoptotic profiles in irradiated patients with increased toxicity

PURPOSE A retrospective study of radiation-induced apoptosis in CD4 and CD8 T-lymphocytes, from 12 cancer patients who displayed enhanced toxicity to radiation therapy and 9 ataxia telangiectasia patients, was performed to test for altered response compared to healthy blood-donors and normal cancer patients. METHODS AND MATERIALS Three milliliters of heparinized blood from each donor was sent via express post to the Paul Scherrer Institute (PSI) for subsequent examination. The blood was diluted 1:10 in RPMI medium, irradiated with 0-, 2-, or 9-Gy X-rays, and incubated for 48 h. CD4 and CD8 T-lymphocytes were then labeled using FITC-conjugated antibodies, erythrocytes were lysed, and the DNA stained with propidium iodide. Subsequently, cells were analyzed using a Becton Dickinson FACScan flow cytometer. Radiation-induced apoptosis was recognized in leukocytes as reduced DNA content attributed to apoptosis-associated changes in chromatin structure. Apoptosis was confirmed by light microscopy, electron microscopy, and by the use of commercially available apoptosis detection kits (in situ nick translation and Annexin V). Data from hypersensitive individuals were compared to a standard database of 105 healthy blood-donors, and a database of 48 cancer patient blood donors who displayed normal toxicity to radiation therapy. To integrate radiosensitivity results from CD4 and CD8 T-lymphocytes after 2 and 9 Gy, z-score analyses were performed. RESULTS A cohort of 12 hypersensitive patients was evaluated; 8 showed enhanced early toxicity, 3 showed enhanced late toxicity, and 1 showed both. The cohort displayed less radiation-induced apoptosis (-1.8 sigma) than average age-matched donors. A cohort of 9 ataxia telangiectasia homozygotes displayed even less apoptosis (-3.6 sigma). CONCLUSION The leukocyte apoptosis assay appears to be a useful predictor of individuals likely to display increased toxicity to radiation therapy; however, validation of this requires a prospective study.

A Versatile and Rapid Assay of Radiosensitivity of Peripheral Blood Leukocytes Based on DNA and Surface-Marker Assessment of Cytotoxicity

We describe a rapid assay to predict intrinsic radiosensitivity of normal tissues based on the radiation-induced cytotoxic response of a number of types of white blood cells. Twenty-four hours after irradiation, leukocytes were prepared for analysis by flow cytometry. Radiation-induced cytotoxicity was characterized by degradation of internucleosomal DNA, which results in a reduced G1/G0-phase DNA peak. Clear differences in the radiosensitivity of five of six different types of leukocytes were observed. Significant increases in cytotoxicity were observed even at 0.5 Gy, indicating the technique may be useful as a biological dosimeter after radiation accidents. Based on data from 12 healthy blood donors, significant inter-donor differences in radiosensitivity were observed after exposure to 2 or 8 Gy of high-dose-rate X rays. Inter-donor variation was significantly greater than intra-donor variation for both CD4 and CD8 T lymphocytes (P < 0.01), and the technique yielded adequate data to stratify donors based on the radiosensitivities of these two cell types alone, requiring less than 1 ml of blood. The assay is a general assay of early cytotoxic response to radiation-induced damage and should be useful when assaying the response to chemotherapy as well.

Sources of variation in patient response to radiation treatment

PURPOSE To investigate sources of variation in radiosensitivity displayed by cancer patients and blood donors using the leukocyte apoptosis assay. METHODS AND MATERIALS Probes were obtained from 105 healthy blood donors, 48 cancer patients displaying normal sensitivity to radiotherapy, 12 cancer patients displaying hypersensitivity to radiotherapy, 12 Ataxia telangiectasia blood donors, and 4 additional individuals with genetic diseases of potentially modified radiosensitivity; 2 neurofibromatosis (NF) donors, a Nijmegen breakage syndrome (NBS) donor, and an Immunodeficiency, Chromosome fragility, Facial anomaly syndrome (ICF) donor. Heparinized blood was diluted in medium, irradiated, and left to incubate for 48 h. CD4 and CD8 T-lymphocyte DNA was stained with propidium iodide and the cells were analyzed by flow cytometry. RESULTS Radiation-induced apoptosis depended on age and cell type. Cohorts of hypersensitive cancer patients, NBS and AT donors displayed compromised apoptotic response. An asymmetric apoptotic response of T-lymphocytes was observed in an ICF donor and a cryptic hypersensitivity donor. Two NF donors displayed no abnormal sensitivity to radiotherapy but compromised apoptotic T-cell response to X-rays. CONCLUSION Our studies reveal 4 physiologic sources of variation in radiation response-2 are genetic: cryptic hypersensitivity and hereditary disease, and 2 are epigenetic: cell type and donor age. They emphasize the important role of proteins involved in the recognition and repair of DNA double-strand breaks in determining the response of individuals to radiotherapy.

Anti‐neuroblastoma antibody chCE7 binds to an isoform of L1‐CAM present in renal carcinoma cells

Immunoprecipitation after cell surface labeling of human neuroblastoma cells showed that the anti‐neuroblastoma monoclonal antibody (mAb) chCE7 binds to a 200,000 Mr cell surface protein. The protein was partially purified by immuno‐affinity chromatography from a human renal carcinoma and a human neuroblastoma cell line, which both showed high levels of binding of MAb chCE7. NH2‐terminal sequences of 18 and 15 amino acid residues were determined. Both sequences isolated from the renal carcinoma and the neuroblastoma cells showed strong homology to human cell adhesion molecule L1 (L1‐CAM), and both were characterized by the NH2‐terminal deletion of 5 amino acids, comprising exon 2 of L1‐CAM. Reverse trancription‐polymerase chain reaction (RT‐PCR) analysis of the regions spanning exon 2 and exon 27 of L1‐CAM indicated that in neuroblastoma cells both transcripts for the full‐length and exon‐deleted forms are present, whereas in the renal carcinoma cell lines only the exon‐deleted L1‐CAM isoform were detected. Western blot analysis showed that 6 of 7 tested renal carcinoma cell lines and 5 of 15 renal carcinoma tissues expressed L1‐CAM. In normal adult kidney tissue, very low levels of protein expression were found. Northern blot analysis confirmed that in renal carcinoma and neuroblastoma cell lines L1‐CAM mRNA levels are correlated with protein expression. Int. J. Cancer 83: 401–408, 1999

m‐THPC‐Mediated Photodynamic Therapy (PDT) Does Not Induce Resistance to Chemotherapy, Radiotherapy or PDT on Human Breast Cancer Cells In Vitro

Photodynamic therapy (PDT) uses laser light to activate a photosensitizer that has been absorbed preferentially by cancer cells after systemic administration. A photo‐toxic reaction ensues resulting in cell death and tissue necrosis. Some cells, however, may survive PDT. This study was performed to determine if surviving human breast cancer cells (MCF‐7) can become resistant to PDT, chemotherapy or radiotherapy. The MCF‐7 cells were cultured under standard conditions prior to being exposed to the photosensitizer, 5,10,15,20‐meta‐tet‐ra(hydroxyphenyl)chlorin (zn‐THPC), for 24 h and then irradiated with laser light (652 nm). Surviving cells were allowed to regrow by allowing a 2 week interval between each additional PDT. After the third and final treatment, colony formation assays were used to evaluate the sensitivity of cultured cells to ionizing radiation and PDT and the ATP cell viability assay tested in vitro chemosen‐sitivity. Flow cytometry was used to analyze the cell cycle. No alterations in the cell cycle were observed after three cycles of PDT with m‐THPC. Similar responses to chemotherapy and ionizing radiation were seen in control and treatment groups. The m‐THPC‐sensitized PDT did not induce resistance to subsequent cycles of PDT, chemo‐ or radiotherapy. Photodynamic therapy with m‐THPC may represent a novel adjunctive treatment of breast cancer that may be combined with surgery, chemotherapy or ionizing radiation.

Staurosporine- and Radiation-Induced G2-Phase Cell Cycle Blocks Are Equally Released by Caffeine

We show here that the arrests of cells in G2 phase of the cell cycle induced by either staurosporine or ionizing radiation are closely related phenomena governed by a common kinase signaling pathway. The protein kinase inhibitor staurosporine induces a complete G2-phase arrest in exponentially growing TK6 human lymphoblastoid and V79 Chinese hamster fibroblast cells. Both cell types are equally sensitive to the kinase inhibitor and the arrest is dependent on its continued presence. Caffeine completely abrogates this arrest at concentrations comparable to those which abrogate radiation-induced G2-phase arrest. The kinetics of caffeine-induced release of both kinds of arrest are essentially identical. The activity of p34cdc2 kinase was also found to increase in a parallel fashion after caffeine-induced release of both kinds of arrest. As opposed to those transformed cell types which arrest only in G2 phase in response to staurosporine, immortalized C3H 10T1/2 fibroblasts and Muntjak skin fibroblasts display both G1- and G2-phase arrests. The results suggest that staurosporine and radiation interact with regulatory pathways in the cell cycle, and specifically with a caffeine-sensitive signal transduction pathway which recognizes DNA damage, regulates the G2/M-phase transition, and attenuates the biological consequences of radiation exposure.

Enhanced neoplastic transformation in an inhomogeneous radiation field: an effect of the presence of heavily damaged cells.

In the inhomogeneous radiation field surrounding small beta-particle sources, nonlethally and heavily damaged cells are in proximity, permitting interaction via extracellular signals. This situation is typical of hot particles such as those released during the accident at Chernobyl. Beta-particle-emitting yttrium-90 wires (average energy 934 keV) were employed to investigate radiation-induced neoplastic transformation under these conditions. Integrated 24-h doses ranging from 0 to 750 Gy across the exposure field were applied. At equal levels of toxicity a 10-fold enhancement of neoplastic transformation frequency in C3H 10T1/2 cells was observed in the presence of heavily damaged cells. Homogeneous fields of low-dose-rate beta-particle radiation produced neoplastic transformation frequencies typical for comparable photon exposures reported in the literature.

Programmed Cellular Response to Ionizing Radiation Damage

Three forms of radiation response were investigated to evaluate the hypothesis that cellular radiation response is the result of active molecular signaling and not simply a passive physicochemical process. The decision whether or not a cell should respond to radiation-induced damage either by induction of rescue systems, e.g. mobilization of repair proteins, or induction of suicide mechanisms, e.g. programmed cell death, appears to be the expression of intricate cellular biochemistry. A cell must recognize damage in its genetic material and then activate the appropriate responses. Cell type is important; the response of a fibroblast to radiation damage is both quantitatively and qualitatively different from that of a lymphocyte. The programmed component of radiation response is significant in radiation oncology and predicted to create unique opportunities for enhanced treatment success.