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In late May of 1903, Troops I and M of the 9th U.S. Cavalry, under the command of Captain Charles Young, left the Presidio in San Francisco, California, for their summer assignment. In sixteen days...

Unresectable pancreatic cancer is almost universally lethal because chemotherapy and radiation cannot completely stop the growth of the cancer. The major problem with using radiation to approximate surgery in unresectable disease is that the radiation dose required to ablate pancreatic cancer exceeds the tolerance of the nearby duodenum. WR-2721, also known as amifostine, is a well-known radioprotector, but has significant clinical toxicities when given systemically. WR-2721 is a prodrug and is converted to its active metabolite, WR-1065, by alkaline phosphatases in normal tissues. The small intestine is highly enriched in these activating enzymes, and thus we reasoned that oral administration of WR-2721 just before radiation would result in localized production of the radioprotective WR-1065 in the small intestine, providing protective benefits without the significant systemic side effects. Here, we show that oral WR-2721 is as effective as intraperitoneal WR-2721 in promoting survival of intestinal crypt clonogens after morbid irradiation. Furthermore, oral WR-2721 confers full radioprotection and survival after lethal upper abdominal irradiation of 12.5 Gy × 5 fractions (total of 62.5 Gy, EQD2 = 140.6 Gy). This radioprotection enables ablative radiation therapy in a mouse model of pancreatic cancer and nearly triples the median survival compared to controls. We find that the efficacy of oral WR-2721 stems from its selective accumulation in the intestine, but not in tumors or other normal tissues, as determined by in vivo mass spectrometry analysis. Thus, we demonstrate that oral WR-2721 is a well-tolerated, and quantitatively selective, radioprotector of the intestinal tract that is capable of enabling clinically relevant ablative doses of radiation to the upper abdomen without unacceptable gastrointestinal toxicity.

Unresectable pancreatic cancer is almost universally lethal because chemotherapy and radiation cannot completely stop the growth of the cancer. The major problem with using radiation to approximate surgery in unresectable disease is that the radiation dose required to ablate pancreatic cancer exceeds the tolerance of the nearby duodenum. WR-2721, also known as amifostine, is a well-known radioprotector, but has significant clinical toxicities when given systemically. WR-2721 is a prodrug and is converted to its active metabolite, WR-1065, by alkaline phosphatases in normal tissues. The small intestine is highly enriched in these activating enzymes, and thus we reasoned that oral administration of WR-2721 just before radiation would result in localized production of the radioprotective WR-1065 in the small intestine, providing protective benefits without the significant systemic side effects. Here, we show that oral WR-2721 is as effective as intraperitoneal WR-2721 in promoting survival of intestinal crypt clonogens after morbid irradiation. Furthermore, oral WR-2721 confers full radioprotection and survival after lethal upper abdominal irradiation of 12.5Gy x 5 fractions (total of 62.5Gy, D2EQ=140.6Gy), which would likely ablate pancreatic cancer. We find that the efficacy of oral WR-2721 stems from its selective accumulation in the intestine, but not in tumors or other normal tissues, as determined by in vivo mass spectrometry analysis. Thus, we demonstrate that oral WR-2721 is a well-tolerated, and quantitatively selective, radioprotector of the intestinal tract that is capable of enabling clinically relevant ablative doses of radiation to the upper abdomen without unacceptable gastrointestinal toxicity.

Purpose To test whether a cyclooxygenase-2 inhibitor (celecoxib) could reduce mortality resulting from radiation-induced pneumonitis. Methods and Materials Celecoxib was given to mice twice daily for 40 consecutive days starting on the day of local thoracic irradiation (LTI) or 40 or 80 days later. C3Hf/KamLaw mice were observed for morbidity, and time to death was determined. Results were analyzed using the Cox proportional hazards model. Results Timing of celecoxib relative to LTI determined efficacy. A significant reduction in time to death was achieved only when celecoxib was started 80 days after LTI, corresponding to the time when pneumonitis is expressed. For these mice the reduction in mortality was quantified as a hazard ratio for mortality of treated vs untreated of 0.36 (95% confidence interval [CI] 0.24-0.53), thus significantly less than 1.0. Correspondingly, the median lethal dose for treated mice (12.9 Gy; 95% CI 12.55-13.25 Gy) was significantly ( P =.026) higher than for untreated mice (12.4 Gy; 95% CI 12.2-12.65 Gy). Conclusions Celecoxib significantly reduced lung toxicity when administered months after LTI when the deleterious effects of radiation were expressed. The schedule-dependent reduction in fatal pneumonitis suggests that celecoxib could be clinically useful by reintroduction of treatment months after completion of radiation therapy. These findings may be important for designing clinical trials using cyclooxygenase-2 inhibitors to treat radiation-induced lung toxicity as a complement to concurrent radiation therapy of lung cancers.

Patients with cancer often experience a high symptom burden prior to the start of treatment. As disease and treatment-related neurotoxicities appear to be additive, targeting disease related symptoms may attenuate overall symptom burden for cancer patients and improve the tolerability of treatment. It has been hypothesized that disease-related symptoms are a consequence of tumor-induced inflammation. We tested this hypothesis using a syngeneic heterotopic murine model of human papilloma virus (HPV)-related head and neck cancer. This model has the advantage of being mildly aggressive and not causing cachexia and weight loss. We previously showed that this tumor leads to increased IL-6, IL-1β, and TNF-α expression in the liver and increased IL-1β expression in the brain. The current study confirmed these features and demonstrated that the tumor itself exhibits high inflammatory cytokine expression (e.g., IL-6, IL-1β, and TNF-α) compared to healthy tissue. While there is a clear relationship between cytokine levels and behavioral deficits in this model, the behavioral changes are surprisingly mild. Therefore, we sought to confirm the relationship between behavior and inflammation by amplifying the effect using a low dose of lipopolysaccharide (LPS, 0.1 mg/kg). In tumor-bearing mice LPS induced deficits in nest building, tail suspension, and locomotor activity approximately 24h after LPS. However, these mice did not display an exacerbation of LPS-induced weight loss, anorexia, or anhedonia. Further, while heightened serum IL-6 was observed there was minimal priming of liver or brain cytokine expression. Next we sought to inhibit tumor-induced burrowing deficits by reducing inflammation using minocycline. Minocycline (~50 mg/kg/day in drinking water) was able to attenuate tumor-induced inflammation and burrowing deficits. These data provide evidence in favor of an inflammatory-like mechanism for the behavioral alterations associated with tumor growth in a syngeneic murine model of HPV-related head and neck cancer. However, the inflammatory state and behavioral changes induced by this tumor clearly differ from other forms of inflammation-induced sickness behavior.

Pancreatic cancer is a lethal disease that is resistant to chemotherapy and radiotherapy. Gemcitabine has recently been shown to be an improvement over 5-fluorouracil in patients with advanced disease. It is also a potent radiosensitizer, which has led to the investigation of gemcitabine with concurrent radiotherapy. However, preliminary results indicate that there are significant limitations to this approach in this challenging disease. Pancreatic cancer cells have alterations in many molecular signaling pathways that may be responsible for their resistance to cytotoxic therapy and aggressive behavior. Cyclooxygenase-2 (COX-2) is commonly overexpressed in pancreatic tumors, and preclinical evidence indicates that selective COX-2 inhibition enhances both chemotherapy and radiotherapy response, without affecting normal tissue damage. We have initiated preclinical studies as well as a phase I clinical protocol evaluating the combination of gemcitabine and celecoxib (Celebrex) with radiotherapy. In preclinical studies, celecelecoxib strongly enhanced the antitumor efficacy of chemoradiation. However, preliminary observations from both the preclinical experiments as well as the clinical protocol have revealed more toxicity with this combination than with gemcitabine and radiotherapy alone. These observations require further study, but are cause for concern when combining gemcitabine, radiotherapy, and celecoxib.

(2002). Out of Fashion: Harriet Hemenway and the Audubon Society, 1896–1905. The Historian: Vol. 65, No. 1, pp. 1-14.

Introduction: Traditional clonogenic survival and high throughput colorimetric assays are inadequate as drug screens to identify novel radiation sensitizers. We developed a method that we call the high content clonogenic survival assay (HCSA) that will allow screening of drug libraries to identify candidate radiation sensitizers. Methods: Drug screen using HCSA was done in 96 well plates. After drug treatment, irradiation, and incubation, colonies were stained with crystal violet and imaged on the INCell 6000 (GE Health). Colonies achieving 50 or more cells were enumerated using the INCell Developer image analysis software. A proof-of-principle screen was done on the KRAS mutant lung cancer cell line H460 and a Custom Clinical Collection (146 compounds). Results: Multiple drugs of the same class were found to be radiation sensitizers and levels of potency seemed to reflect the clinical relevance of these drugs. For instance, several PARP inhibitors were identified as good radiation sensitizers in the HCSA screen. However, there were also a few PARP inhibitors not found to be sensitizing that have either not made it into clinical development, or in the case of BSI-201, was proven to not even be a PARP inhibitor. We discovered that inhibitors of pathways downstream of activated mutant KRAS (PI3K, AKT, mTOR, and MEK1/2) sensitized H460 cells to radiation. Furthermore, the potent MEK1/2 inhibitor tramenitib selectively enhanced radiation effects in KRAS mutant but not wild-type lung cancer cells. Conclusions: Drug screening for novel radiation sensitizers is feasible using the HCSA approach. This is an enabling technology that will help accelerate the discovery of novel radiosensitizers for clinical testing.

We compared and evaluated available laboratory and clinical data on the use of concurrent gemcitabine (Gemzar; Eli Lilly and Company, Indianapolis, IN) and radiation in pancreatic cancer to provide guidance for subsequent prospective research initiatives. Preclinical data suggest that the timing of administration of gemcitabine with respect to radiotherapy is important, but this issue has not yet been confirmed by clinical data. Phase I clinical data indicate that the amount of acute toxicity from the combination of gemcitabine and radiotherapy is strongly related to the dose and schedule of administration of gemcitabine, as well as to the radiation field size. There also appears to be an inverse linear relationship between the maximum tolerated gemcitabine dose and radiation dose. Also important, but less clear, is the infusion rate of gemcitabine as it relates to the systemic efficacy of the drug. The combination of additional agents with gemcitabine and radiation appears to be feasible. Finally, the addition of radioprotectors may enable chemotherapy dose escalation, but safe escalation of the radiotherapy dose with newer techniques has not been established. Semin Oncol 28 (suppl 10):25-33.

We explore the relationship among experimental design, parameter estimation, and systematic error in sloppy models. We show that the approximate nature of mathematical models poses challenges for experimental design in sloppy models. In many models of complex biological processes it is unknown what are the relevant physical mechanisms that must be included to explain system behaviors. As a consequence, models are often overly complex, with many practically unidentifiable parameters. Furthermore, which mechanisms are relevant/irrelevant vary among experiments. By selecting complementary experiments, experimental design may inadvertently make details that were ommitted from the model become relevant. When this occurs, the model will have a large systematic error and fail to give a good fit to the data. We use a simple hyper-model of model error to quantify a model’s discrepancy and apply it to two models of complex biological processes (EGFR signaling and DNA repair) with optimally selected experiments. We find that although parameters may be accurately estimated, the discrepancy in the model renders it less predictive than it was in the sloppy regime where systematic error is small. We introduce the concept of a sloppy system–a sequence of models of increasing complexity that become sloppy in the limit of microscopic accuracy. We explore the limits of accurate parameter estimation in sloppy systems and argue that identifying underlying mechanisms controlling system behavior is better approached by considering a hierarchy of models of varying detail rather than focusing on parameter estimation in a single model., Author Summary Sloppy models are often unidentifiable, i.e., characterized by many parameters that are poorly constrained by experimental data. Many models of complex biological systems are sloppy, which has prompted considerable debate about the identifiability of parameters and methods of selecting optimal experiments to infer parameter values. We explore how the approximate nature of models affects the prospect for accurate parameter estimates and model predictivity in sloppy models when using optimal experimental design. We find that sloppy models may no longer give a good fit to data generated from “optimal” experiments. In this case, the model has much less predictive power than it did before optimal experimental selection. We use a simple hyper-model of model error to quantify the model’s discrepancy from the physical system and discuss the potential limits of accurate parameter estimation in sloppy systems.

Objective: 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. Methods: HNSCC cells and xenografts were used. P16-overexpressing (HPV/p16-negative HN5 and UMSCC-1) and p16 shRNA knockdown (HPV/p16-positive UMSCC-47 and UCPI-SCC154) cells, TRIP12 shRNA or siRNA knockdown (HPV/p16-negative HN5 and Fadu) cells were generated. The effects of p16 or TRIP12 on HNSCC cell radiosensitivity were determined by clonogenic cell survival. The effects of p16 on tumor xenografts (HPV/p16-negative HN5 and HPV/p16-positive UMSCC-47) radioresponse were evaluated by 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 (PCR); and data from The Cancer Genome Atlas HNSCC project were analyzed. Results: P16 expression led to downregulation of TRIP12 protein both in vitro and in vivo via decreasing TRIP12 protein's half life, which in turn led to increased RNF168 levels and subsequently repressed DNA damage repair, represented by increased 53BP1 foci and neutral comet moment tails at 24 hours after irradiation. As a result, p16 expression enhanced radioresponsiveness both in vitro and in vivo. Inhibition of TRIP12 expression led to radiosensitization through repressed DNA damage repair, represented by decreased BRCA1 foci at 1 and 5 hours after irradiation; and increased neutral comet moment tails at 24 hours after irradiation. Furthermore, overexpression of TRIP12 was associated with poor survival in patients with HPV-positive HNSCC. Conclusions: The findings of our study reveal that p16 participates in radiosensitization through influencing DNA damage repair. P16 downregulates TRIP12 protein expression via post-translational regulation. Inhibition of TRIP12 sensitized HNSCC cells via prohibition of DNA damage repair. These findings support the rationale of blocking TRIP12 signaling to improve radiotherapy outcomes. Citation Format: Li Wang, Peijing Zhang, David Molkentine, Jessica Molkentine, Uma Raju, David Valdecanas, Ramesh Tailor, Howard Thames, Thomas Buchholz, Junjie Chen, Li Ma, Kathy Mason, Raymond Meyn, Heath D. Skinner. TRIP12 as a mediator of human papillomavirus/p16-related radiation enhancement effects. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1661.

Background: Head and neck squamous cell carcinoma (HNSCC) tumorigenesis induced by human papillomavirus (HPV) shows a greater clinical response to radiation therapy when compared to HPV negative patients. The underlying mechanism for this more favorable outcome is unknown, but the presumption is that DNA repair response must play a large role. P16INK4a is a known surrogate marker for HPV positivity; however the relationship between radiation sensitivity and P16INK4a has not been completely elucidated in this context. Ubiquitin specific protease 7 (USP7), also known as HAUSP, is a deubiquitylating enzyme that cleaves ubiquitin from substrates to stabilize target proteins. USP7 overexpression is a predictor of poor prognosis in lung carcinomas and correlates with disease progression and lower survival in gliomas. More recent studies have shown USP7 has a role in the stability of DNA damage response proteins such as RNF168, BRCA1 and Chk1; and USP7 knockdown has been shown to radio-sensitize breast cancer cells to radiation. We have investigated the possible role of USP7 in the radio-response of HNSCC cells in the context of HPV status. Methods: Four HPV(+) and four HPV(-) HNSCC were chosen for this study. The radio-response was determined by clonogenic survival assay. P16INK4a overexpression was generated using lentivirus. Western blot analysis was used for visualizing protein expression. The Cancer Genome Atlas was interrogated to examine the relationship between USP7 and clinical outcome. Results: Our data suggest P16INK4a negatively regulates USP7 and leads to the more radiosensitive phenotype associated with HPV(+)HNSCC. Radio-resistant HPV(-) HNSCC cell lines show higher levels of USP7 than HPV(+) lines and over-expression of P16INK4a in HPV(-) HN5 cells leads to down-regulation of USP7 and radio-sensitization. Conversely, P16INK4a siRNA knockdown in HPV(+) UMSCC-47 shows an increase of USP7 protein expression and radio-resistance. P22077, an inhibitor of USP7 activity, was also shown to radiosensitize a HPV(-) cell line. This shows proof of principle that inhibiting USP7 can be a viable approach to sensitizing HPV(-) HNSCC to radiation. USP7 overexpression was also associated with poorer overall survival in HNSCC. Conclusion: These results suggest that USP7 may be a marker of clinical radioresistance in HNSCC and that its inhibition has the potential to improve the treatment outcome of patients with HPV(-) HNSCC when combined with radiotherapy. Citation Format: David Molkentine, Li Wang, Kathleen Bridges, Kathy Mason, Raymond Meyn, Heath D. Skinner. P16INK4a over-expression sensitizes HPV(-) HNSCC to radiation through down-regulation of USP7. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1652.

Background Many prostate cancers demonstrate an increased expression of growth factor receptors such as vascular endothelial growth factor receptor (VEGFR) and platelet derived growth factor receptor (PDGFR) which have been correlated with increased resistance to radiotherapy and poor prognosis in other tumors. Therefore, response to radiation could potentially be improved by using inhibitors of these abnormally activated pathways. We have investigated the radiosensitizing effects of sunitinib, a potent, multi-tyrosine kinase inhibitor of the VEGFR and PDGFR receptors, on human prostate cancer cells. Methods The radiosensitizing effects of sunitinib were assessed on human prostate cancer cell lines DU145, PC3 and LNCaP by clonogenic assay. Sunitinib’s ability to inhibit the activities of its key targets was determined by immunoblot analysis. The radiosensitizing effects of sunitinib in vivo were tested on human tumor xenografts growing in nude mice where response was assessed by tumor growth delay. Results Clonogenic survival curve assays for both DU145 and PC3 cells showed that the surviving fraction at 2 Gy was reduced from 0.70 and 0.52 in controls to 0.44 and 0.38, respectively, by a 24 hr pretreatment with 100 nM sunitinib. LNCaP cells were not radiosensitized by sunitinib. Dose dependent decreases in VEGFR and PDGFR activation were also observed following sunitinib in both DU145 and PC3 cells. We assessed the ability of sunitinib to radiosensitize PC3 xenograft tumors growing in the hind limb of nude mice. Sunitinib given concurrently with radiation did not prolong tumor growth delay. However, when animals were treated with sunitinib commencing the day after fractionated radiation was complete, tumor growth delay was enhanced compared to radiation alone. Conclusions We conclude, based on the in vivo results, that sunitinib and radiation do not interact directly to radiosensitize the PC3 tumor cells in vivo as they did in vitro. The fact that tumor growth delay was enhanced when sunitinib was given after radiotherapy was completed suggests that sunitinib may be acting on the irradiated tumor stroma and suppressing its ability to sustain regrowth of the irradiated tumor. Based on these preclinical findings, we suggest that the combination of sunitinib and radiation for the treatment of prostate cancer deserves further development.

Lung cancer is the leading cause of cancer death in most developed countries. The prognosis remains poor with an overall survival rate at 5 years of only about 15%. Between 70 and 85% of all cases are histologically classified as non-small cell lung carcinoma (NSCLC). Radiation therapy has traditionally been the treatment of choice for locally advanced disease and medically inoperable early stage NSCLC. However radiation therapy alone was not effective treatment for patients with locally advanced NSCLC. The addition of cytotoxic drugs to radiotherapy considerably improves treatment outcome, and the combination of chemotherapy with radiotherapy has become common practice for the treatment of locally advanced lung cancer. The addition of chemotherapy to radiotherapy has two principal objectives: one, to increase the chance of local tumor control and two, to eliminate metastatic disease outside of the radiation field. Several randomized trials have shown improvement of local control and survival by application of concurrent chemotherapy rather than sequential chemotherapy followed by radiation treatment. This combined treatment approach results in median survival times of 13 to 14 months and survival rates at 5 years as high as 15 to 20%. These improvements have been achieved by using standard chemotherapeutic agents, primarily cisplatin-based drug combinations. However, concurrent chemoradiotherapy has increased significant normal tissue toxicity such as esophagitis and pneumonitis. Therefore normal tissue protectors without protective cancer cells became necessary to improve therapeutic ratio. We will discuss mechanism and efficacy of Amifostine to protect normal tissue followed by other normal tissue protectors or molecular targeted treatment without increasing normal tissue damage e.g., prostanoids (COX-2) inhibitors, Growth factor and Cytokines inhibitors, Basic and other inhibitors targeting Fibroblast Growth Factor, Karatinocyte Growth Factors, Epidermoid Growth Factor Receptor, Pentoxifylline, Angiotensin-Converting Enzyme Flavopiridol Poly(ADP-Ribose) Polymerase, Bcl-2 , and Efaproxaril, as well as Radioprotective Gene Therapy/ Antioxidant Therapy: and Superoxide Dismutase

Purpose: C225 strongly enhances tumor radioresponse when given concurrently with radiotherapy. We investigated whether additional therapeutic benefit could be achieved by continuing maintenance treatment with C225 after the completion of fractionated radiotherapy. Methods and Materials: A431 xenografts were treated with local irradiation or combined with C225 by two different schedules: (1) 6 h before the first dose of irradiation and at 3-day intervals for a total of 3 doses during the 7-day fractionated radiotherapy, or (2) 6 doses of C225 given both during radiotherapy and continuing for 3 additional doses after radiotherapy. Tumor cure was assessed by the radiation dose yielding local tumor control in 50% of animals (TCD{sub 50}), and time to recurrence was also determined. Results: Both treatment schedules increased radiocurability as evidenced by reductions in TCD{sub 50}, but the effect was greater when C225 was given both during and after radiotherapy. C225 reduced the TCD{sub 50} of 83.1 (73.2-124.8) Gy by radiation only to 46.2 (39.1-57.5) Gy when given during radiotherapy and to 30.8 (22.2-38.0) Gy when given during and after radiotherapy. Dose modification factors were 1.8 when C225 was given during radiotherapy and 2.7 when given both during and after radiotherapy. C225 was also effective inmore » delaying the onset of tumor recurrences, and was more effective when given as both concurrent and maintenance therapy. Conclusions: Data showed that C225 strongly enhanced the curative effect of fractionated radiation, and its effect was greater if administration was extended beyond the end of radiotherapy. This important finding may influence future designs of clinical trials combining anti-EGFR (anti-epidermal growth factor receptor) agents with radiotherapy.« less

Inhibition of poly(ADP-ribose) polymerase (PARP) by a novel, potent inhibitor, INO-1001, was examined in two rodent and one human fibroblast cell lines, after single and fractionated radiation treatments. Since PARP plays a role in the early events following DNA damage and influences the effectiveness of DNA repair, its inhibition has been proposed to constitute a drug target for the development of novel radiosensitizers. We found that INO-1001 effectively inhibited PARP activity at non-cytotoxic concentrations. Combination treatment of 10 microM INO-1001 and a single dose of radiation resulted in significant radiosensitization of all three cells lines (enhancement ratios 1.4-1.6). This radioenhancement was even greater when the drug and radiation were given as fractionated treatments (enhancement ratio 8.0). Apoptosis (as evaluated by TUNEL staining) was not enhanced by the treatments, suggesting that inhibiting PARP enzyme activity by INO-1001 enhanced radiation-induced cell killing by interfering with DNA repair mechanisms, resulting in necrotic cell death. INO-1001 therefore, appears to have potential as a potent enhancer of radiation sensitivity, without any intrinsic cytotoxicity from the drug alone.

Flavopiridol, a cyclin-dependent kinase (cdk) inhibitor, can cause cell cycle arrest, induce apoptosis in cancer cells, and inhibit tumor cell growth in vivo. The present study investigated the in vitro radiosensitizing effect of flavopiridol and the underlying molecular mechanisms in a murine ovarian cancer cell line, OCA-I. Flavopiridol inhibited cell growth in a dose-dependent manner and enhanced cell radiosensitivity assessed by the clonogenic cell survival assay. A flavopiridol dose of 300 nM, given for 1 day, enhanced radiosensitivity by a factor of 2.1. Clonogenic cell survival after split-dose radiation showed that flavopiridol inhibited repair from radiation damage. In addition, flavopiridol treatment (300 nM, 1 day) resulted in decreased levels of Ku70 and Ku86 proteins that play a role in DNA repair processes, suggesting that DNA repair processes may have been disrupted by this agent. Flow cytometry analysis showed that flavopiridol (300 nM, 1 day) accumulated the cells in G(1) and G(2) phases, with a significant reduction in the S phase component. This cell cycle redistribution is likely another mechanism underlying flavopiridol-induced cell radiosensitivity. Flavopiridol down-regulated cyclin D1 and cyclin E protein levels and also inhibited phosphorylation of retinoblastoma protein, which is inconsistent with the observed cell cycle arrest. Among the cdks tested, cdk-9, the catalytic subunit of positive transcription elongation factor b, was significantly down-regulated by flavopiridol, suggesting that flavopiridol may modulate cellular transcription processes. Furthermore, flavopiridol on its own induced apoptosis in the OCA-I cells, whereas in combination with radiation, exerted no additional increase in apoptosis. Taken together, our data show that flavopiridol strongly augmented the response of ovarian carcinoma cells to radiation and that the underlying mechanisms included inhibition of sublethal DNA damage repair and cell cycle redistribution. At the molecular level, transcriptional regulation by flavopiridol may have been involved.

Human papillomavirus (HPV) type 16 is a major cause of oropharyngeal carcinoma (OPC). P16INK4A has been suggested to be a reliable surrogate marker of HPV-associated OPC with 100% sensitivity and about 80% specificity. Increasing data showed tumor HPV positivity or p16 expression was strongly associated with significantly better prognosis in patients with OPC. As a tumor suppressor gene, p16 has biological functions including regulation of cell cycle progression at the G1/S boundary, angiogenesis, cell senescence, tumor invasion, cell spreading, apoptosis and anoikis. The present study was undertaken to assess the role of p16 in regulating tumor radioresponse and the underlying mechanisms in OPC cell lines. OPC cell lines HN-5 (HPV and p16 negative) and UMSCC-47 (HPV and p16 positive) were used. P16 overexpressing HN-5 cells and shRNA p16 knockdown UMSCC-47 cells were generated using lentivirus vectors. Treatment endpoint was clonogenic cell survival (CSA) determined 10-12 days (for HN5) or 17-20 days (for UMSCC-47) after exposing the cells to 2-10 Gy single doses of γ-radiation (IR). Compared with the control (scramble) cells, p16-overexpressing HN5 cells had significantly higher radiosensitivity (by a factor of 1.56 at 0.1 cell survival fraction); whereas, p16-knockingdown UMSCC-47 cells had less radiosensitivity (by a factor of 1.23 at 0.1 cell survival fraction). Overexpressing P16 in HN-5 cells significantly prolonged the presence of radiation-induced double-strand breaks detected on the basis of 53BP1 foci at 24h after 4 Gy IR. To directly gauge damaged DNA, an alkaline comet assay to detect both single- and double-strand DNA breaks was performed. P16 overexpressing HN-5 cells exhibited a 2.09-fold increase in the comet ‘tail moment’ 48 hours after IR. This finding was supported by increased expression of 53BP1 analyzed by Western blot. In conclusion, other than being a robust surrogate marker for tumor control and survival outcome, our findings demonstrated that p16 also functions as a potent radiation sensitizer. The major underlying mechanism of p16 regulating radiosensitivity is by inhibition of DNA damage repair. Citation Format: Li Wang, Peijing Zhang, David P Molkentine, Hailong Piao, Chunyan Chen, Jessica M Molkentine, Jinsong Zhang, David R Valdecanas, Heath Skinner, Thomas A Buchholz, Junjie Chen, Li Ma, Kathy A Mason, Kie-kian Ang, Raymond E Meyn. P16INK4A, a surrogate marker of HPV infection and prognosis for head and neck cancer, delays DNA damage repair and enhances radiation response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4895. doi:10.1158/1538-7445.AM2014-4895

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.

Docetaxel, a potent chemotherapeutic drug and a strong enhancer of tumor radioresponse, possesses immunomodulating properties. We previously reported that 40% of murine tumors responding to docetaxel by growth delay showed heavy infiltration with macrophages or lymphocytes. The present study explored the effect of whole body irradiation on antitumor action of docetaxel alone or docetaxel plus tumor irradiation. Mice bearing 8-mm MCa-K mammary carcinoma in the leg received 33 mg/kg docetaxel i.v., 5 to 65 Gy tumor irradiation, or both (radiation given 24 h after docetaxel). Docetaxel delayed tumor growth and enhanced the efficacy of radiation: it dramatically reduced TCD50 (radiation dose yielding 50% tumor cure) from the control value of 38.6 Gy to 11.8 Gy, for an enhancement factor of 3.27. In addition to enhancing tumor radioresponse, docetaxel decreased the lung metastatic rate in mice with local tumor control from 26% in mice receiving radiation alone to 11% in mice receiving docetaxel plus radiation. Docetaxel induced heavy infiltration of tumors with lymphocytes, determined 2-4 days after treatment: the percentage of lymphocytes increased from the control value of

(2009). Nature's New Deal: The Civilian Conservation Corps and the Roots of the American Environmental Movement. History: Reviews of New Books: Vol. 37, No. 3, pp. 98-99.

The poly-(ADP-ribose) polymerase (PARP) inhibitor, MK-4827, is a novel potent, orally bioavailable PARP-1 and PARP-2 inhibitor currently in phase I clinical trials for cancer treatment. No preclinical data currently exist on the combination of MK-4827 with radiotherapy (XRT). We examined whether MK-4827 enhances radiosensitivity of a variety of human tumor xenografts of differing p53 status. Human lung cancer xenografts, Calu-6 (p53 null), A549 (p53 wild-type [wt]) and H-460 (p53 wt) and triple negative MDA-MB-231 human breast carcinoma xenografts were used. Tumor xenografts growing in the hind leg of nude mice were exposed to fractionated XRT at 8 mm (diameter). Fractionated XRT (2 Gy per fraction) was delivered for 7 (twice daily, 6 h in between) or 14 (once daily) consecutive days, total dose 28 Gy. MK-4827 was given by gavage once (50 mg/kg, 1 h before first XRT of the day) or twice daily (25 mg/kg, 1 h before each XRT of the day) starting when tumors were 6 mm diameter. Mice received (a) no treatment, (b) MK-4827, (c) XRT or (d) MK-4827 plus XRT. Tumor growth delay (TGD, days for tumors to grow from 8.0 mm to 12.0 mm) was the endpoint. Radiation enhancement factor (EF) was then calculated. MK-4827 alone had no effect on tumor growth for any of the tumors types. For p53 mutant Calu-6 xenografts TGD for untreated tumors was 13.2 ± 0.5 and for twice daily XRT was 30.9 ± 2.4 days. Combination of XRT for 7 days with MK-4827, 25mg/kg twice daily for 21 consecutive days, further extended TGD to 39.7 ± 2.0 days (EF 1.4) and 47.8 ± 4.0 days (EF 2.03) for MK-4827, 50mg/kg once daily. Maximum radiation enhancement was observed when MK-4827 was given at a dose of 50 mg/kg once daily. For XRT and MK-4827 given once daily, MK-4827 was continued until 2 days after completion of XRT, MK-4827 was found to be similarly effective and the EF was 1.7. For p53 wt H460and A459 xenografts, twice daily XRT and once daily MK-4827 was used. MK-4827 was continued until 2 days after completion of XRT. TGD for untreated H460 and A459 xenografts was 7.8 ± 0.5 and 33.2 ± 1.5 days respectively; whereas it was 15.6 ± 0.5 and 53.2 ± 3.1 days respectively for irradiated tumors. TGDs were further extended to 26.0 ± 3.5 days for H460 (EF 2.2) and 64.1 ± 4.2 days (EF 1.9) for A549 xenografts in MK-4827 plus XRT groups. Similarly, using MDA-MB-231 human breast carcinoma xenografts, the potency of combination treatment was greater than XRT alone showing essentially no local tumor regrowth until the occurrence of metastases. When MK-4827 was given as a single dose of 50 mg/kg and tumors were assayed for PAR, MK-4827 reduced PAR levels in tumors by 1 h after administration which persisted for up to 24 h. In conclusion, MK-4827 strongly enhanced the effect of radiation on a variety of human tumor xenografts irrespective of p53 status. The long lasting PARP inhibition (1-24h) potentially adds flexibility to future clinical trial design. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1461. doi:1538-7445.AM2012-1461

(2004). Earth's Magnetism in the Age of Sail. History: Reviews of New Books: Vol. 32, No. 2, pp. 81-81.

There has recently been significant interest in the antitumor activity of anaerobic corynebacteria, potent nonspecific stimulators of the reticuloendothelial system (RES). Corynebacterium parvum was found to be effective against many syngenic and allogenic tumors of experimental animals (for more details, see Milas, 1974). Fewer tumor takes, delay of tumor appearance, and retarded growth were common observations following transplantation of tumor cells in mice treated with this bacterium. Another strain of anaerobic corynebacteria, Corynebacterium granulosum, also proved capable of exerting a strong antitumor adjuvant activity (Milas et al., 1974f; Milas et al., 1974c; Paslin et al., 1974). C. parvum and C. granulosum were found to be potent adjuncts to chemotherapy (Currie and Bagshawe, 1970) and to radiotherapy (to be published) of murine fibrosarcomas. It has recently been observed that complete and lasting regressions of established tumors can be achieved with the intratumor injection of C. parvum (Likhite and Halpern, 1974) or with systemic application of C. parvum or C. granulosum (Milas et al., 1974b; Milas et al., 1974d; Milas et al., 1974a,f).

Corynebacterium parvum given intravenously or into the tumor 7 or 2 days before surgical removal of primary tumors greatly reduced the number of spontaneous pulmonary metastases of a syngeneic mammary carcinoma of C3Hf/Bu mice. When a single dose of 6000 rads γ-rays was used to eliminate primary tumors the number of lung metastases significantly increased. Administration of C. parvum before irradiation not only prevented this metastasis — facilitating effect of radiation, but also reduced the number of lung nodules below that in amputated mice. Metastatic spread was not altered by postoperative treatment of mice with single doses of C. parvum or C. granulosum. However, significantly more mice had lung metastases if they were given 2 intraperitoneal injections of C. granulosum.

A single intraperitoneal (ip) or intravenous (iv) injection of Corynebacterium granulosum into C3Hf/Bu mice shortly after subcutaneous (sc) injection of cells from a strongly antigenic syngeneic fibrosarcoma induced by 3-methylcholanthrene caused complete and lasting regressions of 100 and 70% of resulting tumors, respectively. Treatment with this bacterium sc only slightly inhibited the growth of some tumors. C. granulosum given iv to mice 3 days after the sc injection of fibrosarcoma cells caused complete regressions of 39 of 45 tumors; two iv injections with this immunostimulant given 1 month apart were no more effective than a single injection. Intralesional treatment of fibrosarcomas 8 mm in diameter induced complete regressions of tumors in 30% of the animals, whereas sc treatment contralateral to the growing tumor only slightly reduced tumor growth. Intraperitoneal growth of a fibrosarcoma was efficiently controlled (58-80% survival of mice) if C. granulosum was given ip, but not iv, 3 days after inoculation with tumor cells. Again, two injections of C. granulosum (given ip 4 days apart) were only as effective as a single injection. Treatment with C. granulosum iv at 3, 7, 14, or 21 days after sc inoculation of a weakly antigenic, spontaneously arising mammary carcinoma (MC-1) strongly inhibited tumor growth. Three complete but temporary tumor regressions were observed. The subcutaneous growth of another spontaneous mammary carcinoma (MC-2), which contained fairly strong tumor-specific antigen(s), was also significantly inhibited if C. granulosum was given 3,7, or 14 days after, but not 7 days before, tumor cell inoculation. However, pretreatment of mice with the immunostimulant significantly protected the mice against artifically induced pulmonary metastases of this tumor.