TRIP12 as a mediator of human papillomavirus/p16-related radiation enhancement effects.

Patients with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) have better responses to radiotherapy and higher overall survival rates than do patients with HPV-negative HNSCC, but the mechanisms underlying this phenomenon are unknown. p16 is used as a surrogate marker for HPV infection. Our goal was to examine the role of p16 in HPV-related favorable treatment outcomes and to investigate the mechanisms by which p16 may regulate radiosensitivity. HNSCC cells and xenografts (HPV/p16-positive and -negative) were used. p16-overexpressing and small hairpin RNA-knockdown cells were generated, and the effect of p16 on radiosensitivity was determined by clonogenic cell survival and tumor growth delay assays. DNA double-strand breaks (DSBs) were assessed by immunofluorescence analysis of 53BP1 foci; DSB levels were determined by neutral comet assay; western blotting was used to evaluate protein changes; changes in protein half-life were tested with a cycloheximide assay; gene expression was examined by real-time polymerase chain reaction; and data from The Cancer Genome Atlas HNSCC project were analyzed. p16 overexpression led to downregulation of TRIP12, which in turn led to increased RNF168 levels, repressed DNA damage repair (DDR), increased 53BP1 foci and enhanced radioresponsiveness. Inhibition of TRIP12 expression further led to radiosensitization, and overexpression of TRIP12 was associated with poor survival in patients with HPV-positive HNSCC. These findings reveal that p16 participates in radiosensitization through influencing DDR and support the rationale of blocking TRIP12 to improve radiotherapy outcomes.

Epidermal growth factor receptor and its inhibition in radiotherapy: in vivo findings.

Increasing evidence shows that dysregulated epidermal growth factor receptor (EGFR) signalling plays an important part in neoplasia. When over expressed or mutated, EGFR is frequently associated with more aggressive tumour growth, poor patient prognosis and resistance of tumours to cytotoxic agents, including radiation. The present studies with murine carcinomas showed that there is an inverse correlation between the level of EGFR and tumour radiocurability. Likewise, the present clinical study in patients with head and neck cancer shows that EGFR over expression correlates with poorer tumour response to radiotherapy. Adding EGFR to tumour cells in vitro protected cells against the cytotoxic action of radiation, whereas blocking EGFR with anti-EGFR antibodies enhanced cell radiosensitivity. A casual relationship between EGFR and increased cellular resistance to radiation was established by transferring the EGFR gene into low EGFR-expressing radiosensitive tumour cells, which then become radioresistant. Radiation activated EGFR and its downstream signalling pathways in radioresistant but not in radiosensitive tumours, and this effect was associated with increased resistance to radiation, and enhanced repopulation in irradiated tumours. Increasing evidence shows that blockage of EGFR or interference with any of the steps in its signal transduction cascade can counteract negative outcomes of EGFR signalling, which has recently been explored as a therapeutic strategy in cancer treatment. The present findings demonstrate that treatment of human tumour xenografts with C225, an anti-EGFR monoclonal antibody, dramatically enhanced tumour response to radiation. Overall, the findings show that over expression of EGFR may serve as a predictor of tumour treatment outcome by radiotherapy and as a therapeutic target to enhance the efficacy of radiotherapy.

WR-2721 reduces intestinal toxicity from concurrent gemcitabine and radiation treatment.

BACKGROUND: The nucleoside analog gemcitabine is a potent radiosensitizer of both tumor and normal mucosa, so severe toxic reactions have resulted from its combination with radiation in some clinical treatment schedules for pancreatic cancer. WR-2721 (amifostine) has been shown to reduce normal tissue toxicity produced from both radiation treatment and some chemotherapeutics. The aim of this study was to determine if WR-2721 can protect the gastrointestinal mucosa from injury by concurrent gemcitabine and radiation treatment. METHODS AND MATERIALS: Gemcitabine was injected ip into C3Hf/Kam mice at a concentration of 33 mg/kg 24 h before whole-body irradiation. A single dose (200 mg/kg) of WR-2721 was given 30 min before the radiation treatment or 30 min before gemcitabine or at both times. A quantitative assessment of the chemotherapy/radiation-induced damage was carried out using the mouse microcolony assay for stem cell survival in the intestinal crypts. RESULTS: WR-2721 given 30 min before gemcitabine followed 24 h later by radiation did not confer any protection to the jejunum (DMF 0.95). However, WR-2721 administered 30 min before radiation without or with prior gemcitabine produced protection factors (PF) of 1.35 and 1.42 CONCLUSIONS: WR-2721 did not directly protect the gastrointestinal mucosa from gemcitabine toxicity, but it did protect the gemcitabine-radiosensitized mucosa from acute radiation damage by a factor of 1.42. Therefore, in clinical treatment protocols using concurrent chemoradiation with gemcitabine, WR-2721 may have clinical utility in protecting against radiation-induced mucosal toxicity.

Biology-based combined-modality radiotherapy: workshop report.

PURPOSE: The purpose of this workshop summary is to provide an overview of preclinical and clinical data on combined-modality radiotherapy. METHODS AND MATERIALS: The 8th Annual Radiation Workshop at Round Top was held April 13-16, 2000 at the International Festival Institute (Round Top, TX). RESULTS: Presentations by 30 speakers (from Germany, Netherlands, Australia, England, and France along with U.S. participants and M. D. Anderson Cancer Center faculty) formed the framework for discussions on the current status and future perspectives of biology-based combined-modality radiotherapy. CONCLUSION: Cellular and molecular pathways available for radiation modification by chemical and biologic agents are numerous, providing new opportunities for translational research in radiation oncology and for more effective combined-modality treatment of cancer.

The proliferative response of mouse jejunal crypt cells to radiation-induced cell depletion is not mediated exclusively by transforming growth factor alpha.

Several lines of correlative evidence link transforming growth factor alpha (Tgfa, also known as TGF-alpha) to proliferative activity in jejunal crypt cells. It is therefore tempting to hypothesize that, as a ligand of the epidermal growth factor, it mediates the compensatory proliferative burst in the crypts after radiation-induced cell killing. We have tested this hypothesis by comparing the repopulation response of wild-type and Tgfa-null mice, using the microcolony assay. Mice were exposed whole-body to (137)Cs gamma rays at a dose of approximately 1.6 Gy/min. Single doses and equal doses separated by 4 and 54 h were given. The rightward shift of the dose-response curves for 54 h was identical for wild-type and Tgfa-null mice, and there was no indication of a difference in radiosensitivity. This result indicates that Tgfa is not an essential component of the proliferative response of tissue to radiation-induced cell killing.

A chromosome 15 quantitative trait locus controls levels of radiation-induced jejunal crypt cell apoptosis in mice.

Jejunal crypt cells undergo apoptosis in response to ionizing radiation exposure. In mice the number of cells deleted by apoptosis is determined by several factors including the dose of radiation, the time of day the apoptosis level is quantified, and the strain of mouse irradiated. We previously found that the difference in radiation-induced apoptosis levels between C57BL/6J (B6) and C3Hf/Kam (C3H) mice is controlled by multiple genes, and this set of genes is distinct from that controlling thymocyte apoptosis levels in the same strain combination. Here, we report that a new quantitative trait locus on chromosome 15, Rapop5, partly accounts for the murine strain difference in susceptibility to radiation-induced jejunal crypt cell apoptosis. In addition, we show sexual dimorphism in the extent of radiation-induced jejunal crypt cell apoptosis, with female mice having higher levels.

Evolution and development of pigment cells: at the crossroads of the discipline.

The following is a summary of the current state of comparative biology with respect to pigmentation. Recent results from molecular analyses of genes involved in pigmentation in lower vertebrates are compared with similar data from mouse and man. Particular emphasis has been placed on evolutionary and developmental aspects of pigmentation. Recent advances in molecular biology of lower vertebrate pigmentation allow for the comparison of orthologous molecules across a wider range of species than ever before; some of these results are summarized and used to highlight the current state of pigmentation from a comparative perspective. A more cellular, organismal approach is also explored to highlight some important lessons from comparative biology. Lastly, large-scale evolutionary questions are put into a framework that highlights both the differences and similarities between mammals/birds and other vertebrates. It is the opinion of the authors that important, long-standing questions in these areas can now be addressed in ways that have not been possible before. Thus, the discipline is at an exciting crossroads where developmental and evolutionary data can be used to create a unified view of pigment cells and pigments across many species.

Nuclear scintigraphic assessment of radiation-induced intestinal dysfunction.

Radiation-induced damage to the intestine can be measured by abnormalities in the absorption of various nutrients. Changes in intestinal absorption occur after irradiation because of loss of the intestinal absorptive surface and a consequent decrease in active transport. In our study, the jejunal absorption of (99m)Tc-pertechnetate, an actively transported gamma-ray emitter, was assessed in C3H/Kam mice given total-body irradiation with doses of 4, 6, 8 and 12.5 Gy and correlated with morphological changes in the intestinal epithelium. The absorption of (99m)Tc-pertechnetate from the intestinal lumen into the circulation was studied with a dynamic gamma-ray-scintigraphy assay combined with a multichannel analyzer to record the radiometry data automatically in a time-dependent regimen. The resulting radioactivity-time curves obtained for irradiated animals were compared to those for control animals. A dose-dependent decrease in absorptive function was observed 3.5 days after irradiation. The mean absorption rate was reduced to 78.8 +/- 9.3% of control levels in response to 4 Gy total-body irradiation (mean +/- SEM tracer absorption lifetime was 237 +/- 23 s compared to 187 +/- 12 s in nonirradiated controls) and to 28.3 +/- 3.7% in response to 12.5 Gy (660 +/- 76 s). The decrease in absorption of (99m)Tc-pertechnetate at 3.5 days after irradiation correlated strongly (P < 0.001) with TBI dose, with the number of cells per villus, and with the percentage of cells in the crypt compartment that were apoptotic or mitotic. A jejunal microcolony assay showed no loss of crypts and hence no measured dose-response effects after 4, 6 or 8 Gy TBI. These results show that dynamic enteroscintigraphy with sodium (99m)Tc-pertechnetate is a sensitive functional assay for rapid evaluation of radiation-induced intestinal damage in the clinically relevant dose range and has a cellular basis.

[Inhibition of tumor neo-angiogenesis and induction of apoptosis as properties of docetaxel (taxotere)]. / Hemmung der Tumorneoangiogenese und Induktion von Apoptose als Eigenschaften von Docetaxel (Taxotere).

Paclitaxel and docetaxel are potent drugs that are effective in the treatment of malignant tumors. The cytotoxic action of these drugs is not fully understood, but it appears to be mediated mainly through mitotic arrest and subsequent apoptosis. Because no information is available on the antiangiogenesis action of docetaxel, the investigations were performed to determine whether inhibition of neoangiogenesis plays a role in docetaxel's antitumor efficacy. Four different mouse tumors, two squamous cell carcinomas (SCC-IV; SCC-VII) and two adenocarcinomas (MCA-4; MCA-29) were assayed for angiogenic activity using the in vivo i.c. angiogenesis assay. Tumor cells (5 x 10(5)) were injected i.c. into the skin flap over the abdominal wall, and the number of new blood vessels at the tumor cell injection site was determined 2, 4, 6, 8, 10 and 12 days later. The mice were treated with docetaxel (Taxotere--31.3 mg/kg i.v.) 1 or 4 days after tumor cell injection. The number of new blood vessels increased with time. Docetaxel reduced the number of newly formed blood vessels in MCAs, but not in SCCs. The reduction was associated with slower tumor growth. In a separate set of experiments we observed that docetaxel's inhibitory effect on the two MCAs was histologically associated with massive tumor cell destruction by means of both apoptosis and necrosis. This was not observed for the two SCCs. Since no reduction in blood vessels occurred in tumors unresponsive to docetaxel, the inhibition of neoangiogenesis in docetaxel-responsive tumors was likely the result of a decrease in angiogenic stimuli due to docetaxel's destruction of tumor cells.

Maximizing therapeutic gain with gemcitabine and fractionated radiation.

PURPOSE/OBJECTIVE: The nucleoside analogue gemcitabine inhibits cellular repair and repopulation, induces apoptosis, causes tumor growth delay, and enhances radiation-induced growth delay. After single doses of drug and radiation, maximum enhancement of tumor response was obtained when gemcitabine preceded radiation by at least 24 h. Conversely, the cellular radioresponse of the normal gastrointestinal epithelium was slightly protected when gemcitabine and radiation were separated by 24 h. This differential response created a time frame within which therapeutic gain could be maximized. In our present investigation, we sought to define the most therapeutically beneficial scheme of gemcitabine administration when combined with fractionated radiotherapy. METHODS AND MATERIALS: C3Hf/Kam mice were given identical drug and radiation schedules of administration, and both normal tissue (jejunal mucosa) and tumor (Sa-NH) responses were measured. Irradiation was given once per day for 5 days in normal tissue and tumor growth delay studies and twice per day for the tumor cure endpoint. A total dose of 25 mg/kg gemcitabine was given i.p. in 1 of 3 schedules: a single dose of 25 mg/kg 24 h before the start of fractionated irradiation, 12.5 mg/kg 24 h before the first and third radiation doses, or 24 h before each of 5 radiation doses. Groups of mice bearing 7- or 8-mm diameter tumors were treated with gemcitabine alone or in combination with fractionated irradiation under ambient or hypoxic conditions. The survival response of the jejunal mucosa was quantified by the microcolony assay and histologically by quantifying apoptosis, mitosis, S-phase fraction, and crypt cellularity. RESULTS: For tumor growth delay, dose-modifying factors (DMFs) were similar (1.34-1.46) for all 3 schedules of drug administration. In contrast, the response of the jejunum was strongly dependent on the schedule of gemcitabine administration. A single dose of gemcitabine before the start of fractionated radiotherapy resulted in slight radioprotection (DMF 0.96). Two doses and 5 daily doses of gemcitabine enhanced radiation response by factors of 1.09 and 1.23, respectively. Major factors affecting the response of the jejunal mucosa were apoptotic death of S-phase cells exposed to gemcitabine and cell cycle synchrony of surviving cells. Tumor reoxygenation was found to be a major mechanism for tumor radioenhancement, in addition to those reported earlier. CONCLUSION: All 3 schedules of drug administration produced therapeutic gain; however, when gemcitabine was given more than once in a 5-fraction radiation treatment schedule, normal tissue toxicity increased. The highest therapeutic gain (1.4) was achieved by giving a single dose of gemcitabine (25 mg/kg) 24 h before the start of fractionated radiotherapy.

Combination of taxanes with radiation: preclinical studies.

Treatment regimens consisting of taxanes, a potent class of chemotherapeutic agents, combined with radiotherapy have recently undergone extensive preclinical investigation. The focus was primarily on cell radiosensitization because taxanes arrest cells in the radiosensitive G2/M phase of the cell cycle. In vitro studies provided ample evidence that taxanes can enhance radiation sensitivity of tumor cells, with enhancement factors ranging from 1. 1 to more than 3.0. Additive or subadditive effects were also reported. The outcome of the taxane-radiation interaction in vitro depended on many factors, including cell type, proliferation state of cells, drug concentration, and timing of radiation delivery in relation to drug administration. In vivo studies, although limited, showed that taxanes can strongly enhance tumor radioresponse, producing enhancement factors of 1.2 to more than 2.0. Two major mechanisms of tumor radioenhancement were detected: reoxygenation of radioresistant hypoxic cells and G2/M arrest. Both occur in tumors that respond to taxanes by mitotic arrest and apoptosis. Only G2/M arrest occurs in tumors that display only mitotic arrest. Compared with tumor radioresponse, normal tissue radioresponse was much less affected by taxanes. On a molecular level, taxanes activate a number of genes, but it appears that their effects are mainly p53-independent and primarily involve phosphorylation of the Bcl-2 gene. Overall, preclinical studies show that taxanes can enhance radiation sensitivity of tumor cells, potentiate tumor response, and increase the therapeutic ratio of radiotherapy. The cellular and molecular effects of taxanes may be useful in designing optimal treatment schedules for clinical trials.

Lack of correlation between mitotic arrest or apoptosis and antitumor effect of docetaxel.

PURPOSE: To determine, as we did for paclit-axel, whether mitotic arrest and apoptosis induced in murine tumors in vivo by docetaxel correlate with the drug's antitumor effect and whether the antitumor efficacy of docetaxel depends on p53 mutational status of tumors. METHODS: C3Hf/Kam mice were implanted with one of the following 15 syngeneic tumors: seven adenocarcinomas (MCa-4, MCa-29, MCa-35, MCa-K, OCa-I, ACa-SG, and HCa-I), two squamous cell carcinomas (SCC-IV and SCC-VII), five sarcomas (FSa, FSa-II, Sa-NH, NFSa, and Sa-4020) and one lymphoma (Ly-TH). When the tumors had grown to 8 mm in diameter, the mice were treated with 31.3 mg/kg docetaxel i.v. Tumor growth delay was the endpoint of docetaxel's antitumor effect. In separate groups of mice, mitotic arrest and apoptosis were determined micromorphometrically 1 to 72 h after docetaxel treatment. Tumors were assayed for their p53 status by sequence analysis of RNA prepared from freshly excised tumors. RESULTS: Docetaxel caused statistically significant growth delay in six of seven adenocarcinomas, three of five sarcomas, and the lymphoma, but not in either of the squamous cell carcinomas. The drug induced mitotic arrest in all tumor types, but to various degrees ranging from 6.4+/-0.4% to 25.1+/-0.1%. In contrast, docetaxel induced appreciable apoptosis in only 5 of 15 tumors, with 10.3+/-1.6% being the highest apoptotic value. Neither mitotic arrest nor apoptosis were significantly correlated with tumor growth delay. However, tumors that responded to docetaxel by significant tumor growth delay histologically displayed massive cell destruction by cell lysis, and four of these tumors also showed marked infiltration with mononuclear lymphoid cells. Of the 15 tumors only 3 had mutant p53. CONCLUSIONS: Docetaxel exhibited a strong antitumor effect in two-thirds of murine tumors, and on a milligram per kilogram basis was more effective than paclitaxel against the same tumors. The drug was a potent inducer of mitotic arrest but a weak inducer of apoptosis, neither of which correlated with its antitumor effect. Tumor cell lysis appeared to be a major mode of tumor cell destruction and can be regarded as the main mechanism underlying antitumor efficacy of docetaxel. In contrast, paclitaxel's antitumor efficacy is related to its ability to induce apoptosis. At the molecular level, there was no dependency of antitumor efficacy of docetaxel on p53 mutational status of tumors.

Effect of docetaxel on the therapeutic ratio of fractionated radiotherapy in vivo.

The aim of this investigation was to determine whether docetaxel increases the therapeutic ratio of fractionated radiotherapy in vivo. Two tumor types were chosen based on their sensitivity to docetaxel as a single agent: (a) docetaxel-sensitive MCa-4 mammary adenocarcinoma, which responds to docetaxel by G2-M-phase cell cycle arrest, apoptosis, and subsequent reoxygenation of surviving tumor cells; and (b) docetaxel-resistant SCC-VII squamous cell carcinoma, which responds to docetaxel treatment only by G2-M-phase arrest. Response of the normal jejunal mucosa in mice was compared to the response of both tumor types to confirm therapeutic gain. We conducted micromorphometric analysis of tumor cell mitosis, assayed apoptosis by its histological appearance in tissue sections, and determined tumor response by tumor growth delay. Normal tissue response of the jejunum was assayed by micromorphometric analysis of mitotic and apoptotic indices, and clonal crypt stem cell survival was measured using the microcolony assay. Two clinically relevant treatment schedules were tested for both antitumor efficacy and normal tissue toxicity: (a) a single bolus of docetaxel (33 mg/kg i.v.) 24 h before five daily fractions of radiation; and (b) daily administration of docetaxel (8 mg/kg i.v.) with radiation delivered at the peak of mitotic arrest (9 h for MCa-4 and 6 h for SCC-VII tumors). The best therapeutic gain for docetaxel-sensitive MCa-4 was achieved with a single bolus of drug 24 h before the start of fractionated radiotherapy (therapeutic gain = 2.04). This schedule takes advantage of reoxygenation of hypoxic tumor cells during the interval between drug treatment and radiation delivery. The best therapeutic gain for docetaxel-resistant SCC-VII was achieved with intermittent multiple doses of docetaxel given during the course of fractionated radiotherapy. This schedule maximized the exposure of cells to radiation while they were arrested by docetaxel in the radiosensitive G2-M phases of the cell cycle (enhancement factor = 2.0). Final therapeutic gain was reduced to 1.59 because of increased normal tissue toxicity in mice treated with multiple intermittent doses of docetaxel in combination with fractionated radiotherapy. Thus, docetaxel greatly enhanced tumor response to fractionated radiotherapy, but the magnitude of therapeutic efficacy depended on drug-radiation scheduling. The greatest therapeutic gain in the treatment of docetaxel-sensitive tumors was achieved by a single large dose of docetaxel administered 1 day before the initiation of fractionated radiotherapy and in the treatment of docetaxel-resistant tumors by daily concomitant docetaxel-radiation treatments.

A non-parametric method of reconstructing single-dose survival curves from multi-fraction experiments.

PURPOSE: A method of estimating the single-dose curve from designed multifraction experiments is described and applied to three datasets. MATERIALS AND METHODS: The method, which is non-parametric and based on standard statistical regression techniques, can be used for functional endpoints which are either continuous or binary. The datasets are concerned with wound healing on mice, myelopathy in guinea pigs and spermatogenesis in mice. The results are compared with the results from fitting the linear quadratic model. The statistical methods of Bootstrapping and residual plots are illustrated. RESULTS: The method is based in part on an assumed statistical model, however, exact knowledge of the correct statistical model is not necessary to obtain an estimate of the shape of the single-dose survival curve. We find no good evidence from the reconstructed single-dose survival curve of an "induced repair" phenomena at low doses for the wound healing and spermatogenesis experiments. For the myelopathy experiment the data are consistent with the LQ model with a low alpha-beta ratio down to doses of at least 1.5 Gy per fraction. CONCLUSIONS: A robust statistical method of estimating the shape of the single-dose survival curve is demonstrated using standard statistical software on three datasets.

Induction of transforming growth factor alpha in irradiated mouse jejunum.

PURPOSE: To determine the involvement of the mitogenic growth factors transforming growth factor alpha (TGF alpha), epidermal growth factor (EGF), and the EGF receptor (EGF-R) in the proliferative response after irradiation of the mouse jejunum. METHODS AND MATERIALS: C3Hf/Kam mice were whole-body irradiated with 5 and 11 Gy 250 kV X rays. Mice were killed 1-10 days after irradiation, and immunohistochemistry, in situ hybridization (ISH), and RNase protection assays were performed. RESULTS: Damage to the jejunal crypts caused by irradiation resulted in a strong proliferative response 1-5 days after 5 Gy and 3-6 days after 11 Gy. Expression of TGF alpha, EGF, and EGF-R increased at 1-2 days and decreased at 4-8 days after 5- or 11-Gy irradiation. Also, TGF alpha mRNA increased during the early phase of the proliferative response (1-2 days after 5 or 11 Gy) followed by a decrease at 4 days after 5 Gy and 8 days after 11 Gy. CONCLUSION: These data indicate that, at the beginning of the proliferative response after irradiation, the transcription of TGF alpha mRNA is increased, and that it is inhibited just before compensatory proliferation decreases. Thus, active regulation of TGF alpha expression takes place at least at the transcriptional level, resulting in upregulation of TGF alpha production and increased TGF alpha levels in the crypts during the proliferative response.