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Squamous cell carcinoma driven by human papillomavirus (HPV) is more sensitive to DNA-damaging therapies than its HPV-negative counterpart. Here, we show that p16, the clinically used surrogate for HPV positivity, renders cells more sensitive to radiotherapy via a ubiquitin-dependent signaling pathway, linking high levels of this protein to increased activity of the transcription factor SP1, increased HUWE1 transcription, and degradation of ubiquitin-specific protease 7 (USP7) and TRIP12. Activation of this pathway in HPV-positive disease led to decreased homologous recombination and improved response to radiotherapy, a phenomenon that can be recapitulated in HPV-negative disease using USP7 inhibitors in clinical development. This p16-driven axis induced sensitivity to PARP inhibition and potentially leads to “BRCAness” in head and neck squamous cell carcinoma (HNSCC) cells. Thus, these findings support a functional role for p16 in HPV-positive tumors in driving response to DNA damage, which can be exploited to improve outcomes in both patients with HPV-positive and HPV-negative HNSCC. Significance: In HPV-positive tumors, a previously undiscovered pathway directly links p16 to DNA damage repair and sensitivity to radiotherapy via a clinically relevant and pharmacologically targetable ubiquitin-mediated degradation pathway.

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Supplementary Data from p16 Represses DNA Damage Repair via a Novel Ubiquitin-Dependent Signaling Cascade

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Squamous cell carcinoma driven by human papillomavirus (HPV) is more sensitive to DNA-damaging therapies than its HPV-negative counterpart. Here, we show that p16, the clinically used surrogate for HPV positivity, renders cells more sensitive to radiotherapy via a ubiquitin-dependent signaling pathway, linking high levels of this protein to increased activity of the transcription factor SP1, increased HUWE1 transcription, and degradation of ubiquitin-specific protease 7 (USP7) and TRIP12. Activation of this pathway in HPV-positive disease led to decreased homologous recombination and improved response to radiotherapy, a phenomenon that can be recapitulated in HPV-negative disease using USP7 inhibitors in clinical development. This p16-driven axis induced sensitivity to PARP inhibition and potentially leads to “BRCAness” in head and neck squamous cell carcinoma (HNSCC) cells. Thus, these findings support a functional role for p16 in HPV-positive tumors in driving response to DNA damage, which can be exploited to improve outcomes in both patients with HPV-positive and HPV-negative HNSCC.Significance:In HPV-positive tumors, a previously undiscovered pathway directly links p16 to DNA damage repair and sensitivity to radiotherapy via a clinically relevant and pharmacologically targetable ubiquitin-mediated degradation pathway.

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Squamous cell carcinoma driven by human papillomavirus (HPV) is more sensitive to DNA-damaging therapies, such as radiation, than its HPV-negative counterpart. Here we show that p16, the clinically utilized surrogate for HPV positivity, renders cells more sensitive to radiation via a ubiquitin-dependent signaling pathway, linking high levels of this protein to increased activity of the transcription factor SP1, increased HUWE1 transcription and degradation of ubiquitin-specific protease 7 (USP7). Activation of this pathway in HPV-positive disease leads to an absence of TRIP12, decreased DNA damage repair, improved response to radiation and better clinical outcomes. Conversely, repression of this pathway in HPV-negative disease is druggable via USP7 inhibitors under clinical development, resulting in potentiation of radiation response. Our findings may lead to improved outcomes for patients with HPV-negative radioresistant tumors, while allowing decreased intensity of therapy for patients with HPV-positive tumors.

Human papillomavirus (HPV) drives the development of squamous cell carcinoma at several sites, including the oropharynx. Generally, the presence of HPV renders a tumor more sensitive to DNA-damaging therapies such as radiation; however, the mechanism behind this phenomenon is elusive. Previous studies have shown that p16, the clinically utilized surrogate for HPV tumor positivity, can render cells more sensitive to radiation. In the current manuscript, using a combination of immunoprecipitation mass spectrometry (IP/MS), in vivo and in vitro modulation and clinical tumor profiling, we identify a novel ubiquitin-dependent signaling pathway linking p16 to increased activity of the transcription factor SP1 leading to increased HUWE1 transcription and degradation of ubiquitin-specific protease 7 (USP7). This pathway is activated in HPV-positive tumor cells, leading to an absence of TRIP12, decreased DNA damage repair and increased mitotic death following radiation. As USP7 inhibitors are currently in clinical trials, this pathway provides a novel means by which radioresistant tumors may be targeted to increase response and improve outcome.

PURPOSE: Head and neck cancers (HNSCC) are routinely treated with radiotherapy; however normal tissue toxicity remains a concern. Therefore, it is important to validate treatment modalities combining molecularly targeted agents with radiotherapy to improve the therapeutic ratio. The aim of this study was to assess the ability of the PARP inhibitor niraparib (MK-4827) alone, or in combination with cell cycle checkpoint abrogating drugs targeting Chk1 (MK-8776) or Wee1 (MK-1775), to radiosensitize HNSCCs in the context of HPV status. MATERIALS AND METHODS: Accepted Manuscript PARP1, PARP2, Chk1 or Wee1 shRNA constructs were analyzed from an in vivo shRNA screen of HNSCC xenografts comparing radiosensitization differences between HPV(+) and HPV(−) tumors. Radiosensitization by niraparib alone or in combination with MK-8776 or MK-1775 was assessed by clonogenic survival in HPV(−) and HPV(+) cells; and the role of p16 in determining response was explored. Relative expressions of DNA repair genes were compared by PCR array in HPV(+) and HPV(−) cells, and following siRNA-mediated knockdown of TRIP12 in HPV(−) cells. RESULTS: In vivo shRNA screening showed a modest preferential radiosensitization by Wee1 and PARP2 in HPV(−) and Chk1 in HPV(+) tumor models. Niraparib alone enhanced the radiosensitivity of all HNSCC cell lines tested. However, HPV(−) cells were sensitized to a greater degree, as suggested by the shRNA screen. When combined with MK-8776 or MK-1775, radiosensitization was further enhanced in an HPV dependent manner with HPV(+) cells enhanced by MK-8776 and HPV(−) cells enhanced by MK-1775. A PCR array for DNA repair genes showed PARP and HR proteins BRCA1 and RAD51 were much lower in HPV(+) cells than in HPV(−). Similarly, directly knocking down p16-dependent TRIP12 decreased expression of these same genes. Overexpressing p16 decreased TRIP12 expression and increased radiosensitivity in HPV(−) HN5. However, while PARP inhibition led to significant radiosensitization in the control, it led to no further significant radiosensitization in p16 overexpressing cells. Forced p16 expression in HPV(−) HN5 increased accumulation in G1 and subG1 and limited progression to S phase, thus reducing effectiveness of PARP inhibition. CONCLUSIONS: Niraparib effectively radiosensitizes HNSCCs with a greater benefit seen in HPV(−). HPV status also plays a role in response to MK-8776 or MK-1775 when combined with niraparib due to differences in DNA repair mechanisms. This study suggests that using cell cycle abrogators in combination with PARP inhibitors may be a beneficial treatment option in HNSCC, but also emphasizes the importance of HPV status when considering effective treatment strategies.

Anthracycline chemotherapy (AC) is associated with decline in left ventricular ejection fraction (LVEF), yet the mechanisms remain unclear. Although changes in microRNAs (miRs) have been identified in adult cardiovascular disease, miR profiles in pediatric patients with AC have not been well studied. The goal of this study was to examine miR profiles (unbiased array) in pediatric patients with AC compared with age-matched referent normal patients. We hypothesize that pediatric patients with AC will express a unique miR profile at the initiation and completion of therapy and will be related to LVEF. Serum was collected in pediatric patients (10–22 yr, n = 12) with newly diagnosed malignancy requiring AC within 24–48 h after the initiation of therapy (30–60 mg/m2) and ~1 yr after completing therapy. A custom microarray of 84 miRs associated with cardiovascular disease was used (quantitative RT-PCR) and indexed to referent normal profiles (13–17 yr, n = 17). LVEF was computed by cardiac MRI. LVEF fell from AC initiation at ~1 yr after AC completion (64.28 ± 1.78% vs. 57.53 ± 0.95%, respectively, P = 0.004). Of the 84 miRs profiled, significant shifts in 17 miRs occurred relative to referent normal ( P ≤ 0.05). Moreover, the functional domain of miRs associated with myocardial differentiation and development fell over threefold at the completion of AC ( P ≤ 0.05). Moreover, eight miRs were significantly downregulated after AC completion in those patients with the greatest decline in LVEF (≥10%, P < 0.05). This study demonstrates, for the first time, that changes in miR expression occur in pediatric patients with AC. These findings suggest that miRs are a potential strategy for the early identification of patients with AC susceptible to left ventricular dysfunction. NEW & NOTEWORTHY Although anthracycline chemotherapy (AC) is effective for a number of pediatric cancers, an all too often consequence of AC is the development of left ventricular failure. The present study identified that specific shifts in the pattern of microRNAs, which regulate myocardial growth, function, and viability, occurred during and after AC in pediatric patients, whereby the magnitude of this shift was associated with the degree of left ventricular failure.

The present study was undertaken to explore the possible mechanisms of the behavioral alterations that develop in response to cancer and to cancer therapy. For this purpose we used a syngeneic heterotopic mouse model of human papilloma virus (HPV)-related head and neck cancer in which cancer therapy is curative. Mice implanted or not with HPV+ tumor cells were exposed to sham treatment or a regimen of cisplatin and radiotherapy (chemoradiation). Sickness was measured by body weight loss and reduced food intake. Motivation was measured by burrowing, a highly prevalent species specific behavior. Tumor-bearing mice showed a gradual decrease in burrowing over time and increased brain and liver inflammatory cytokine mRNA expression by 28 days post tumor implantation. Chemoradiation administered to healthy mice resulted in a mild decrease in burrowing, body weight, and food intake. Chemoradiation in tumor-bearing mice decreased tumor growth and abrogated liver and brain inflammation, but failed to attenuate burrowing deficits. PCR array analysis of selected hypoxia and mitochondrial genes revealed that both the tumor and chemoradiation altered the expression of genes involved in mitochondrial energy metabolism within the liver and brain and increased expression of genes related to HIF-1α signaling within the brain. The most prominent changes in brain mitochondrial genes were noted in tumor-bearing mice treated with chemoradiation. These findings indicate that targeting mitochondrial dysfunction following cancer and cancer therapy may be a strategy for prevention of cancer-related symptoms.

We have collected lab notebooks from Rod Wither's many years of experimentation, from laboratories in Houston and Los Angeles, as well as from several of his collaborators in the USA and overseas. The contents have been digitized, and in this note we explain the mechanism that has been set up to make the 'Withers Archive' available online.

Interaction between the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGF-1R) has been well established in many cancer types. We investigated the effects of cetuximab (EGFR antibody) and IMC-A12 (IGF-1R antibody) on the response of head and neck squamous cell carcinoma (HNSCC) to radiation therapy (RT). The effects of cetuximab and IMC-A12 on cell viability and radiosensitivity were determined by clonogenic cell survival assay. Formation of nuclear γ-H2AX and 53BP1 foci was monitored by immunofluorescence. Alterations in target signaling were analyzed by Western blots. In vivo tumor growth delay assay was performed to determine the efficacy of triple therapy with IMC-A12, cetuximab, and RT. In vitro data showed that cetuximab differentially affected the survival and the radiosensitivity of HNSCC cells. Cetuximab suppressed DNA repair that was evident by the prolonged presence of nuclear γ-H2AX and 53BP1 foci. IMC-A12 did not have any effect on the cell survival. However, it increased the radiosensitivity of one of the cell lines. EGFR inhibition increased IGF-1R expression levels and also the association between EGFR and IGF-1R. Addition of IMC-A12 to cetuximab did not increase the radiosensitivity of these cells. Tumor xenografts exhibited enhanced response to RT in the presence of either cetuximab or IMC-A12. Concurrent treatment regimen failed to further enhance the tumor response to cetuximab and/or RT. Taken together our data suggest that concomitant inhibition of both EGFR and IGF-1R pathways did not yield additional therapeutic benefit in overcoming resistance to RT.

Dr. Rodney Withers (“Rod”) died in Houston, TX, on 25 February 2015 at the age of 82 after a long struggle with a variant of Parkinson's disease. He was a strong supporter of the Radiation Research...

Purpose: To test the appropriateness of the linear-quadratic (LQ) model to describe survival of jejunal crypt clonogens after split doses with variable (small 1–6 Gy, large 8–13 Gy) first dose, as a model of its appropriateness for both small and large fraction sizes. Methods: C3Hf/KamLaw mice were exposed to whole body irradiation using 300 kVp X-rays at a dose rate of 1.84 Gy/min, and the number of viable jejunal crypts was determined using the microcolony assay. 14 Gy total dose was split into unequal first and second fractions separated by 4 h. Data were analyzed using the LQ model, the lethal potentially lethal (LPL) model, and a repair-saturation (RS) model. Results: Cell kill was greater in the group receiving the larger fraction first, creating an asymmetry in the plot of survival vs size of first dose, as opposed to the prediction of the LQ model of a symmetric response. There was a significant difference in the estimated bs (higher b after larger first doses), but no significant difference in the as, when large doses were given first vs small doses first. This difference results in underestimation (based on present data by approximately 8%) of isoeffect doses using LQ model parameters based on small fraction sizes. While the LPL model also predicted a symmetric response inconsistent with the data, the RS model results were consistent with the observed asymmetry. Conclusion: The LQ model underestimates doses for isoeffective crypt-cell survival with large fraction sizes (in the present setting, >9 Gy).

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. The current study examined combined treatment efficacy of MK-4827 and fractionated radiotherapy using a variety of human tumor xenografts of differing p53 status: Calu-6 (p53 null), A549 (p53 wild-type [wt]) and H-460 (p53 wt) lung cancers and triple negative MDA-MB-231 human breast carcinoma. To mimic clinical application of radiotherapy, fractionated radiation (2 Gy per fraction) schedules given once or twice daily for 1 to 2 weeks combined with MK-4827, 50 mg/kg once daily or 25 mg/kg twice daily, were used. MK-4827 was found to be highly and similarly effective in both radiation schedules but maximum radiation enhancement was observed when MK-4827 was given at a dose of 50 mg/kg once daily (EF = 2.2). MK-4827 radiosensitized all four tumors studied regardless of their p53 status. MK-4827 reduced PAR levels in tumors by 1 h after administration which persisted for up to 24 h. This long period of PARP inhibition potentially adds to the flexibility of design of future clinical trials. Thus, MK-4827 shows high potential to improve the efficacy of radiotherapy.

Introduction Anthracycline chemotherapy (AC) can be associated with a progressive decline in left ventricular ejection fraction (LVEF), but the contributory mechanisms remain unclear. While changes in microRNAs (miRs), a key translational control point, have been identified in adult cardiovascular disease, whether and to what degree a shift in miR profiles occurs in pediatric AC patients has not been well studied. The goal of this study was to examine miR profiles (unbiased array) in pediatric AC patients compared to age matched referent normal patients. Hypothesis We hypothesize that pediatric AC patients will express a unique miR profile at initiation and completion of therapy and will be related to LVEF. Methods Serum was collected in pediatric patients (n=12, 13-22 years old) with newly diagnosed malignancy requiring AC within 24-48 hours following initiation of therapy (30-60 mg/m2) and approximately one year after completing maximal therapy. A custom microarray of 84 miRs associated with cardiovascular disease pathways was utilized (RT-qPCR) and indexed to referent normal profiles (n=17, 13-17 years old). LVEF was computed by cardiac MRI. Results LVEF fell from AC initiation at one year following AC (64.28% ± 1.78% vs 57.53% ± 0.95% respectively, p=0.004). Of the 84 miRs profiled, significant shifts in 17 miRs occurred relative to referent normal (p ≤ 0.05) where a nearly equivalent number were upregulated and downregulated (41 vs 59%, respectively). Moreover, the functional domain of miRs associated with myocardial differentiation and development fell by over 3-fold at completion of AC (Figure, p ≤ 0.05). Lastly, miRs-143-3p, -499a-5p, and -142-3p were associated with the fall in LVEF (p ≤ 0.05). Conclusions This study demonstrated for the first time that dynamic changes in miR expression occurs in pediatric cancer patients following AC. These findings suggest that miRs are a potential strategy for early identification of patients most susceptible to LV dysfunction following AC exposure, as well as potential targets for protective therapies.

Poly(ADP-ribose) synthetase inhibitor, INO-1001, is known to sensitize cells to radiation in vitro by inhibiting the repair of DNA damage. Recent evidence has suggested that PARP inhibition may also be a way of selectively targeting p53 deficient cancer cells. The present study tested INO-1001 for its in vivo effect on the chemoresponse of two p53 deficient tumors, human breast cancer MDA-MB-231 and murine mammary carcinoma MCa-K. Doxorubicin was used as the DNA damaging agent and tumor growth delay assay was used as the endpoint. Results showed that INO-1001 was highly effective in enhancing the anti-tumor effects of Doxorubicin for both MDA-MB-231 (EF = 1.88) and MCa-K (EF = 1.64). We conclude that PARP inhibitor INO-1001 has high potential for enhancing the anti-tumor effects of chemotherapy agents such as Doxorubicin against p53 deficient breast cancer.

Purpose: 130-nm albumin–bound paclitaxel (nab-paclitaxel) is a novel solvent-free albumin-bound paclitaxel, designed to avoid solvent-related toxicity. Nab-paclitaxel has been successfully introduced into the clinic but its radiation-enhancing potential has not yet been evaluated. We conducted a preclinical evaluation of the radiation-modulating effects of nab-paclitaxel in tumor and normal tissues. Experimental Design: Mice bearing syngeneic ovarian or mammary carcinomas were treated with nab-paclitaxel, radiation, or combination of both. Nab-paclitaxel was administered at 90 mg/kg, 1.5 times the maximum tolerated dose for solvent-based paclitaxel. End points were antitumor efficacy (growth delay, radiocurability, and cellular effects) and normal tissue toxicity (gut and skin). Results: Nab-paclitaxel showed single-agent antitumor efficacy against both tumor types and acted as a radiosensitizer. Combined with radiation, nab-paclitaxel produced supra-additive effects when given before radiation. Nab-paclitaxel significantly increased radiocurability by reducing the dose yielding 50% tumor cure (TCD50) from 54.3 to 35.2 Gy. Tumor histology following nab-paclitaxel treatment was characterized by pronounced necrotic and apoptotic cell death and mitotic arrest. Nab-paclitaxel did not increase normal tissue radioresponse. Conclusions: Nab-paclitaxel exhibited strong antitumor efficacy against both tumors as a single agent and it improved radiotherapy in a supra-additive manner. These improved effects were achieved without increased normal tissue toxicity to either rapidly or slowly proliferating normal tissues although the drug dose was 1.5 times higher than the maximum tolerated dose of solvent-based paclitaxel. These preclinical findings show that combining nab-paclitaxel with radiotherapy would improve the outcome of taxane-based chemoradiotherapy. This novel taxane is thus a good candidate for testing in clinical chemoradiotherapy trials.

Despite recent improvements in chemotherapy and radiation therapy in cancer management with the addition of biological agents, novel treatment approaches are needed to further benefit patients. Cyclo-oxygenase (COX)-2 inhibition represents one such possibility. COX-2 is an enzyme induced in pathological states such as inflammatory disorders and cancer, where it mediates production of prostanoids. The enzyme is commonly expressed in both premalignant lesions and malignant tumours of different types. A growing body of evidence suggests an association of COX-2 with tumour development, aggressive biological tumour behaviour, resistance to standard cancer treatment, and adverse patient outcome. COX-2 may be related to cancer development and propagation through multiple mechanisms, including stimulation of growth, migration, invasiveness, resistance to apoptosis, suppression of the immunosurveillance system, and enhancement of angiogenesis. Epidemiological data suggest that NSAIDs and selective COX-2 inhibitors might prevent the development of cancers, including colorectal, oesophageal and lung cancer. Preclinical investigations have demonstrated that inhibition of this enzyme with selective COX-2 inhibitors enhances tumour response to radiation and chemotherapeutic agents. These preclinical findings have been rapidly advanced to clinical oncology. Clinical trials of the combination of selective COX-2 inhibitors with radiotherapy, chemotherapy or both in patients with a number of cancers have been initiated, and preliminary results are encouraging. This review discusses the role of COX-2, its products (prostaglandins) and its inhibitors in tumour growth and treatment.

Purpose Recently we reported that inhibition of cyclin-dependent kinases (cdks) by flavopiridol enhanced the radiation response of murine ovarian carcinoma cells in culture. The purpose of this investigation was to extend these studies to in vivo tumor models and test whether flavopiridol increases the therapeutic ratio of radiotherapy. Methods and materials Three transplantable syngeneic mouse tumors were used: mammary carcinoma (MCa-29), ovarian carcinoma (OCa-I), and a lymphoma (Ly-TH). Tumor treatment endpoints included growth delay, cure, and spontaneous lung metastases (OCa-I tumor). The normal tissue endpoint was survival of jejunal crypt cells quantified microscopically. A range of flavopiridol doses from 0.625 to 5.0 mg/kg were given systemically once or twice daily over 5, 10, or 20 days. Combined therapy flavopiridol treatments were initiated either several days before or shortly after the start of single dose or daily fractionated radiotherapy. Results The major findings of this study are that all three tumors treated with flavopiridol alone responded by tumor growth delay. Two of the tumors (MCa-29 and Ly-TH) responded in a schedule-dependent manner with larger radiation enhancement factors when flavopiridol treatment was started a few hours after irradiation (radioenhancement factors [EF] Ly-TH = 2.04, EF MCa-29 = 1.50 for single dose irradiation). When combined with fractionated irradiation (2.6 Gy daily for 10 or 20 days), flavopiridol enhanced the response of the MCa-29 tumor by a factor of 1.25–1.46. A fractional radiation dose of 6 Gy in combination with flavopiridol produced a 62.5% cure rate compared with 25% tumor cure for radiation alone. A novel finding of this study was the demonstration of antimetastatic activity of flavopiridol in addition to its effect on the local primary tumor. Both the incidence and absolute number of lung metastasis were reduced when flavopiridol followed surgical removal of the large (10 mm) primary leg tumor. The normal jejunum treated with flavopiridol and radiation responded in a schedule independent manner and the degree of radioenhancement (EF, 1.05–1.06) was much less than for any of the tumors studied. Conclusions Therapeutic gain was achieved when flavopiridol treatment was initiated either before or after the start of radiotherapy. Flavopiridol shows promising clinical potential administered alone or in combination with other cytotoxic agents, including both chemotherapy and radiotherapy.

Cyclooxygenase-2 (COX-2) is an enzyme expressed primarily in pathologic states, such as inflammatory disorders and cancer, where it mediates prostaglandin production. Its overexpression is associated with more aggressive biologic tumor behavior and adverse patient outcome. Increasing evidence shows that agents that selectively inhibit COX-2 enhance tumor response to radiation or chemotherapeutic agents. This article gives an overview of some of this evidence. In addition, we describe new results showing that celecoxib, a selective COX-2 inhibitor, enhanced response of A431 human tumor xenografts in nude mice to radiation by an enhancement factor (EF) of 1.43 and to the chemotherapeutic agent docetaxel by an EF of 2.07. Celecoxib also enhanced tumor response when added to the combined docetaxel plus radiation treatment (EF = 2.13). Further experiments showed that selective COX-2 inhibitors enhanced tumor cell sensitivity to ionizing radiation, involving inhibition of cellular repair from radiation damage and cell cycle redistribution as mechanisms for some cell types. The results show that selective COX-2 inhibitors have the potential to improve tumor radiotherapy or radiochemotherapy, and this therapeutic strategy is currently under clinical testing.

Purpose: Conjugating drugs with polymeric carriers is one way to improve selective delivery to tumors. Poly (L-glutamic acid)-paclitaxel (PG-TXL) is one such conjugate. Compared with paclitaxel, its uptake, tumor retention, and antitumor efficacy are increased. Initial studies showed that PG-TXL given 24 h before or after radiotherapy enhanced tumor growth delay significantly more than paclitaxel. To determine if PG-TXL-induced enhancement is obtained in a more clinically relevant setting, we investigated PG-TXL effects on tumor cure. Methods and Materials: Mice bearing 7-mm-diameter ovarian carcinomas were treated with PG-TXL at an equivalent paclitaxel dose of 80 mg/kg, single dose or 5 daily fractions of radiation or both PG-TXL and radiation. Treatment endpoint was TCD 50 (radiation dose yielding tumor control in 50% of mice). Acute radioresponse of jejunum, skin, and hair was determined for all treatments. Results: PG-TXL dramatically improved tumor radioresponse, reducing TCD 50 of single-dose irradiation from 53.9 (52.2–55.5) Gy to 7.5 (4.5–10.7) Gy, an enhancement factor (EF) of 7.2. The drug improved the efficacy of fractionated irradiation even more, reducing the TCD 50 of 66.6 (62.8–90.4) Gy total fractionated dose to only 7.9 (4.3–11.5) Gy, for an EF of 8.4. PG-TXL did not affect normal tissue radioresponse resulting from either single or fractionated irradiation. Conclusion: PG-TXL dramatically potentiated tumor radiocurability after single-dose or fractionated irradiation without affecting acute normal tissue injury. To our knowledge, PG-TXL increased the therapeutic ratio of radiotherapy more than that previously reported for other taxanes, thus, PG-TXL has a high potential to improve clinical radiotherapy.

Positive transcription elongation factor-b (P-TEFb) is a complex containing CDK9 and a cyclin (T1, T2 or K). The effect of inhibition of P-TEFb by 5,6-dichloro-l-β-D-ribofuranosyl benzimidazole (DRB) on cell radiosensitivity and the underlying mechanisms were investigated.Six human cancer cell lines were subjected to (3)H-uridine incorporation, cell viability and clonogenic cell survival assays; cell-cycle redistribution and apoptosis assay; western blots and nuclear 53BP1 foci analysis after exposing the cells to DRB with/without γ-radiation.DRB suppressed colony formation and enhanced radiosensitivity of all cell lines. DRB caused a further increase in radiation-induced apoptosis and cell-cycle redistribution depending on p53 status. DRB prolonged the presence of radiation-induced nuclear p53 binding protein-1 (53BP1) foci and suppressed the expression of sirtuin-1 (SIRT1) and casein kinase 2-alpha (CK2α), suggesting an inhibition of DNA repair processes.Our findings indicate that DRB has the potential to increase the efficacy of radiotherapy and warrants further investigation using in vivo tumor models.

Purpose To investigate differences in tumor histotype, incidence, latency, and strain susceptibility in mice exposed to single-dose or clinically relevant, fractioned-dose γ-ray radiation. Methods and Materials C3Hf/Kam and C57BL/6J mice were locally irradiated to the right hindlimb with either single large doses between 10 and 70 Gy or fractionated doses totaling 40 to 80 Gy delivered at 2-Gy/d fractions, 5 d/wk, for 4 to 8 weeks. The mice were closely evaluated for tumor development in the irradiated field for 800 days after irradiation, and all tumors were characterized histologically. Results A total of 210 tumors were induced within the radiation field in 788 mice. An overall decrease in tumor incidence was observed after fractionated irradiation (16.4%) in comparison with single-dose irradiation (36.1%). Sarcomas were the predominant postirradiation tumor observed (n=201), with carcinomas occurring less frequently (n=9). The proportion of mice developing tumors increased significantly with total dose for both single-dose and fractionated schedules, and latencies were significantly decreased in mice exposed to larger total doses. C3Hf/Kam mice were more susceptible to tumor induction than C57BL/6J mice after single-dose irradiation; however, significant differences in tumor susceptibilities after fractionated radiation were not observed. For both strains of mice, osteosarcomas and hemangiosarcomas were significantly more common after fractionated irradiation, whereas fibrosarcomas and malignant fibrous histiocytomas were significantly more common after single-dose irradiation. Conclusions This study investigated the tumorigenic effect of acute large doses in comparison with fractionated radiation in which both the dose and delivery schedule were similar to those used in clinical radiation therapy. Differences in tumor histotype after single-dose or fractionated radiation exposures provide novel in vivo evidence for differences in tumor susceptibility among stromal cell populations.

Polymer complex constructed from WR 2721 and poly(hydroxyoxyethylene phosphate) was synthesized. The structure of complex formed was elucidated by 1H-, 13C, 31P NMR and FT-IR spectroscopy. The radioprotector was immobilized via ionic bonds. Radioprotective efficacy was evaluated by clonal survival of stem cells in crypts of mouse small intestine, and incidence and latency of the acute radiation induced bone marrow syndrome. Protection factors were assessed for WR 2721 and for the polymer complex. Protection factors for the polymer complex ranged from 2.6 for intestinal stem cell survival to 1.35 for 30 day survival (LD50) following whole body radiation exposure. In all cases, the polymer complex was a significantly better radiation protector than the parent compound.

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.

Evans, S. C., Mack, D. C., Mason, K. A. and Thames, H. D. The Proliferative Response of Mouse Jejunal Crypt Cells to Radiation-Induced Cell Depletion is not Mediated Exclusively by Transforming Growth Factor Alpha. Radiat. Res. 155, 864–867 (2001). Several lines of correlative evidence link transforming growth factor alpha (Tgfa, also known as TGF-α) 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 137Cs γ 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 ...

Prostaglandins are arachidonate metabolites produced in virtually all mammalian tissues and possess diverse biologic capabilities, including vasoconstriction, vasodilatation, stimulation or inhibition of platelet aggregation, and immunomodulation, primarily immunosupression (1–4). They are implicated in the promotion of development and growth of malignant tumors (4–7). They are also involved in the response of tumor and normal tissues to cytotoxic agents such as ionizing radiation (8). Prostaglandin production is mediated by two cyclooxygenase enzymes: cyclooxygenase-1 and cyclooxygenase-2. Cyclooxygenase-1 is constitutively expressed and is ubiqui tous , and cyclooxygenase-2 is induced by diverse inflammatory stimuli (7,9). Nonsteroidal anti-inflammatory drugs (NSAIDs) or agents inhibit cyclooxygenase enzymes and consequently can prevent, inhibit, or abolish the effects of prostaglandins. Increasing evidence shows that NSAIDs can inhibit the development of cancer in both experimental animals and in humans (7), can reduce the size of established tumors (6–8), and can increase the efficacy of cytotoxic anticancer agents (8). Our own investigations have demonstrated that the NSAID indomethacin prolongs tumor growth delay and increases the tumor cure rate in mice after radiotherapy (8,10,11). Commonly used NSAIDs, including indomethacin, inhibit both cyclooxygenase-1 and cyclooxygenase-2. However, treatment with these agents may be limited by toxicity to normal tissue, particularly by ulcerations and bleeding in the gastrointestinal tract ascribed to the inhibition of cyclooxygenase-1. Recently developed selective cyclooxygenase-2 inhibitors exert potent anti-inflammatory activity but cause fewer unwanted side effects (7,9,12,13). These compounds may thus be safer than those NSAIDs that are in common use. A recent report (7) shows that cyclooxygenase-2-specific inhibitors can prevent carcinogenesis in experimental animals, but their efficacy in enhancing in vivo tumor response to radiation has not been established. By use of the mouse sarcoma NFSA, we investigated the potential of 4-[5-(4chlorophenyl)-3-(trifluoromethyl)-1Hpyrazol-l-yl]benzenesulfonamide (SC8236), a selective cyclooxygenase-2 inhibitor (14,15) (supplied by Searle, G. D. & Co., Skokie, IL), to enhance response of tumor to local g-irradiation. All studies reported had institutional approval and all guidelines for appropriate animal treatment were followed. We have reported earlier (6) that the NFSA sarcoma is a nonimmunogenic and prostaglandin-producing tumor that spontaneously developed in C3Hf/Kam mice. This tumor exhibits an increased radioresponse if indomethacin is given prior to tumor irradiation (10,11). In experiments described in this communication, solitary tumors were generated in the right hind legs of mice by the injection of 3 × 10 viable NFSA tumor cells. When tumors were 8 mm in diameter, they were locally irradiated with 25–80 Gy single-dose g-radiation. Treatment with SC-8236 (6 mg/kg body weight, given in the drinking water) was started when tumors were approximately 6 mm in diameter, and the treatment was continued for 10 consecutive days. In some experiments, tumor irradiation was performed 3–8 days after initiation of the treatment with SC-8236. The end points of the treatment were tumor growth delay (days) and TCD50 (tumor control dose 50, defined as the radiation dose yielding local tumor cure in 50% of irradiated mice 120 days after irradiation). Treatment of mice with SC-8236 alone significantly inhibited tumor growth (inset in Fig. 1, A). Tumor diameter doubling time, based on tumor growth from 6 to 12 mm in diameter, was increased from 7.3 days (95% confidence interval [CI] 4 6.4–8.1 days) to 14.8 days (95% CI 4 11.5–18.1 days) (P

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.

Purpose: The study investigated whether basal, constitutive levels of p21 WAF1/CIP1 protein in murine carcinomas are related to in vivo tumor radioresponse. The study is based on recent observations demonstrating that in vitro cancer cell lines are resistant to cytotoxic drugs when they express high basal levels of p21 WAF1/CIP1 protein, and that the loss of the p21 gene in the HCT116 human colorectal cancer cell line results in increased radioresponse of xenografts derived from that cell line. Methods and Materials: Protein levels of p21 WAF1/CIP1 , p53, bax, and bcl-2 were determined in 8 carcinomas (3 mammary carcinomas designated MCa-4, MCa-29, and MCa-35, 2 squamous cell carcinomas designated SCC-IV and SCC-VII, ovarian adenocarcinoma OCa-I, hepatocarcinoma HCa-I, and adenosquamous carcinoma ACa-SG) syngeneic to C3Hf/Kam mice using Western blot analysis. The tumors, growing in the right hind legs of mice, were 8 mm in diameter at the time of analysis. These tumors greatly differ in their radioresponse, assessed by TCD50 assay, and in their susceptibility to radiation-induced apoptosis. Results: Protein levels of these oncogenes varied among tumors, with p21 WAF1/CIP1 showing the greatest variation: its mean densitometric value ranged from 1 to 19. Bcl-2 levels also showed broad variation in densitometric values, from 1 to 10. In comparison, bax and p53 (7 of 8 tumors contained wild-type p53) varied much less among different tumor types; their variation was within a 5-fold range, and the level of p53 was similar in 6 of 8 tumors. Tumor radioresponse correlated significantly (R = 0.77, p = 0.02) only with the magnitude of p21 WAF1/CIP1 expression: tumors with high levels of p21 WAF1/CIP1 were less radiocurable than those with lower levels. Tumor radiocurability showed a significant positive correlation ( p = 0.02) with the extent of radiation-induced apoptosis, indicating that tumors that responded to radiation with higher percentages of apoptosis were more curable by radiation. Despite a strong trend to correlation, ( p = 0.15), p21 WAF1/CIP1 expression did not correlate significantly with radiation-induced apoptosis, which suggested that p21 WAF1/CIP1 influenced tumor radioresponse by mechanisms beyond that of apoptosis induction. Conclusion: Our findings showed that murine tumors exhibit wide variation in constitutive levels of p21 WAF1/CIP1 which had a significant relationship with tumor radioresponse: tumors with high levels of p21 WAF1/CIP1 were less radiocurable than those with lower levels. These findings support the concept that p21 WAF1/CIP1 is a major determinant of tumor radioresponse in vivo , and may have important clinical implications. The pretreatment assessment of p21 WAF1/CIP1 protein could serve as a useful predictor of radiotherapy outcome and may assist in selecting an effective treatment modality.

The clinical use of protons for the treatment of cancer is one of the most technologically advanced forms of therapy. The newly commissioned M.D. Anderson Cancer Center, Proton Therapy Center Houston is one of the newest facilities offering a complete range of advanced proton therapies designed specifically to minimize treatment-related normal tissue toxicity. As an integral part of the clinical commissioning of this proton beam, we performed an in vivo assessment of the relative biologic effectiveness of the 250-MeV protons relative to standard cobalt 60 using the jejunal crypt microcolony assay in mice. Our data are consistent with a generic relative biologic effectiveness of 1.1, and this relative biologic effectiveness is in agreement with that used to describe most other clinical proton therapy beams worldwide.

As cancer-related symptoms are similar to those of sickness associated with inflammation, it has been proposed that they are also mediated by increased inflammatory cytokines in the brain. However, there is still limited evidence for this relationship. The current study evaluated the alternative possibility that cancer-related symptoms are associated with changes in gene expression indicative of hypoxia signaling and mitochondrial abnormalities using PCR Arrays. Mice were injected or not with heterotopic tonsil epithelial cells transfected with Ras, E6/E7 of HPV16 and were exposed or not to 3 rounds of cisplatin (5.28 mg/kg) and radiotherapy (8 Gy). Tumor-bearing mice showed increased brain and liver inflammatory cytokine mRNA expression that was associated with decreased burrowing. Chemoradiation in non-tumor-bearing mice decreased burrowing but did not induce liver or brain inflammatory cytokine expression. Chemoradiation in tumor-bearing mice reduced burrowing and attenuated liver and brain inflammation, presumably by reducing tumor volume. Both the tumor and its treatment were associated gene expression changes indicative of mitochondrial dysfunction and HIF-1 α activation in liver and brain, with evidence of an additive effect of tumor and chemoradiation on brain mitochondrial dysfunction. Although correlative at this stage, these findings indicate that targeting mitochondrial dysfunction following cancer and cancer therapy may be a strategy for prevention of cancer-related symptoms. Ongoing work is exploring whether this can be achieved without interfering with the tumor response to chemoradiation.

A method of estimating the single-dose curve from designed multifraction experiments is described and applied to three datasets.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.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.A robust statistical method of estimating the shape of the single-dose survival curve is demonstrated using standard statistical software on three datasets.

This article reports data that describe perturbations in cell-cycle dynamic kinetic parameters following in vivo treatment of a syngeneic murine mammary tumor (MCa-4) with paclitaxel. The kinetic parameters include the duration of the S (Ts) and G2M (TG2M) phases of the cell cycle, the tumor potential doubling time (Tpot), the bromodeoxyuridine (BrdUrd) labeling index (Ll), and the fraction of proliferating cells that successfully completed mitosis following paclitaxel treatment. When tumors transplanted into the right thigh of 4-month-old male C3Hf/kam mice grew to 8 mm mean diameter, the mice were injected intravenously with 40 mg/kg paclitaxel. At one of a range of times thereafter (1–72 hours) animals were pulse labeled with 60 mg/kg BrdUrd. Mice were sacrificed and tumors excised either immediately or at 3 or 6 hours after BrdUrd labeling. Single-nuclei suspensions were prepared, and the kinetic parameters evaluated using bivariate DNA versus BrdUrd flow-cytometric methods. For at least 72 hours after paclitaxel treatment, the major cell kinetic perturbation was a consistent prolongation of the duration of G2M from approximately 2 hours to 5 hours. Tpot increased from about 30 hours to 40 hours. The rate of cell progression through S-phase remained unchanged. The fraction of BrdUrd-labeled cells that completed mitosis was markedly reduced by paclitaxel (to 10% of control values when measured 7 hours after treatment) but never reached zero. Apoptotic nuclei were discernible as BrdUrd-labeled particles with sub-G1 DNA contents. These data show that in this model tumor system, paclitaxel treatment leads to profound cell-cycle perturbations, largely a buildup of cells in G2M. This phenomenon is expected to contribute to the radiosensitization of this tumor by paclitaxel, in addition to reoxygenation that we reported earlier (1).

Tumor necrosis and oxygen status were investigated as a function of tumor size in three syngeneic murine carcinomas, MCa-4, OCa-I, and SCC-VII, in C3Hf/Kam mice. Tumor necrosis was estimated histologically, and tumor oxygenation determined by direct polarographic histography. As tumor volume increased necrosis increased significantly in all three tumor types (p < 0.001). Similarly, as tumor volume increased from 200 to 1400 mm3, hypoxia, defined as the percentage of measured pO2 values < or = 5.0 mm Hg, increased from 55.1% to 95.9%, 70.3% to 81.4%, and 56.8% to 98.5% in MCa-4, OCa-I, and SCC-VII tumors respectively (p < 0.001). Correcting pO2 for necrosis reduced the tumor size dependence of measured tumor hypoxia in all three tumor types but in no case was the reduction significant. The main effect of correction was to shift the fitted curves of percent pO2 values < or = 5.0 mm Hg down toward lower percentages for all tumors. This change was significant for MCa-4 and OCa-1 tumors (p < 0.001), but not for SCC-VII (p = 0.054). Defining the influence of variables such as necrosis that affect polarographic assessment of tumor oxygenation is important to enhance the technique's reliability and prospect as an investigative and predictive tool.

Background : Microtubules are cellular organelles with functions that include control of cell division by mitosis, cell morphology, and transport of material within the cell. The anticancer drug paclitaxel (Taxol) promotes accelerated assembly of excessively stable microtubules. Consequently, treated cells tend to become arrested in mitosis. The drug also induces apoptotic cell death in vitro and in vivo. Prior to this study, the relative contributions of mitotic arrest and apoptosis to the in vivo antitumor effect and the relationship between the two factors had not been established ; moreover, it is not known whether paclitaxel-induced mitotic arrest inevitably results in cell death. Purpose : Our aim was to quantify the mitotic arrest and apoptosis induced by paclitaxel in 16 murine tumors in vivo and to correlate these two factors with the drug's antitumor effect. Methods : Inbred C3Hf/Kam mice were implanted with one of the following 16 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), six sarcomas (FSa, FSa-II, Sa-IIa, Sa-NH, NFSa, and Sa-4020), and one lymphoma (Ly-TH). The tumor growth delay induced by paclitaxel (40 mg/kg body weight given intravenously) was measured in 163 control and 163 treated mice, and its significance was assessed by Student's t test. In a separate group of 439 mice, the percentage of cells in mitosis or apoptosis was scored micromorphometrically at various times after paclitaxel administration. The significance of correlations between paclitaxel-induced tumor growth delay and paclitaxel-induced levels of mitosis or apoptosis was determined by simple correlation and Spearman's rank correlation. P values reported represent two-sided tests of statistical significance. Results : Statistically significant tumor growth delays were found in response to paclitaxel treatment of mice for three of four murine mammary carcinomas (all P≤.010), an ovarian carcinoma (P =.00003), a salivary gland adenocarcinoma (P = .0002), a lymphoma (P =.0002), and two of six sarcomas (both P≤.034), but not for either of two squamous cell carcinomas or for the hepatocellular carcinoma. Paclitaxel-induced mitotic arrest was apparent in all tumor types, but to various degrees, and was not significantly correlated with growth delay (R 2 =.16 ; P =.124). In contrast, apoptotic cell death in response to paclitaxel was not ubiquitous, but it was strongly correlated with growth delay (R 2 =.59 ; P =.001). The pretreatment level of apoptosis was correlated with both paclitaxel-induced apoptosis (R 2 =.71 ; P =.00004) and tumor growth delay (R 2 =.55 ; P =.001). Conclusion : The antitumor effect of paclitaxel was correlated with paclitaxel-induced apoptosis and base-line apoptosis, but not with mitotic arrest. Implications : Apoptosis is an important mechanism of cell death in response to paclitaxel treatment of in vivo murine tumors. An underlying tumor type-specific propensity for apoptosis is implied by the correlation between pretreatment and paclitaxel-induced apoptosis. Both the extent of pretreatment apoptosis and the paclitaxel-induced percentage of apoptosis may be useful predictors of response to the drug.

The tumor suppressor gene p53 maintains the integrity of the genome by stimulating apoptosis in cells that have sustained DNA damage. The p53 gene is frequently altered in human cancers, including breast cancer, and such alterations are thought to result in genomic instability and aneuploidy, 2 hallmarks of anaplastic cells. We used radiation as a DNA-damaging agent to test the role of p53 in controlling apoptosis propensity in pre-neoplastic mammary lesions in mice. Four different pre-neoplastic mammary outgrowth lines, D1, TM2H, TM4 and TM12, were maintained by serial transplantation in the cleared mammary fat pads of syngeneic BALB/c mice. These lines have known alterations in p53 expression : TM12 has normal expression, D1 over-produces wild-type protein, TM2H contains a deletion resulting in a null phenotype and TM4 produces a mutant protein. Mice bearing the various outgrowths were irradiated with 5 Gy, and apoptosis was scored by examination of histological sections prepared from the outgrowths 6 hr after irradiation. The TM 12 outgrowths, but not the TM2H outgrowths, exhibited radiation-induced apoptosis. The D1 and TM4 lines expressed radiation-induced apoptosis while the fat pads were being repopulated ; however, the induced apoptosis declined to low levels as the mice aged. These results are consistent with the hypothesis that normal p53 function is important if mammary cells with DNA damage are to be deleted by apoptosis.

Levels of radiation-induced jejunal crypt cell apoptosis were compared in C57BL/6J, C3Hf/Kam and C3H/HeJ mice. Apoptosis levels were consistently lower in the C3H strains than in C57BL/6J. Although other explanations are possible, the strain difference is most likely to have a genetic basis, and in fact a preliminary analysis of the F2 progeny of C3H/HeJ and C57BL/6J mice indicates that more than one gene is involved. Both C3H strains also had lower levels of radiation-induced thymocyte apoptosis than C57BL/6J mice. Jejunal crypt cell apoptosis levels did not co-segregate with thymocyte apoptosis levels in the F2 progeny of C57BL/6J and C3H/HeJ mice. These results imply that the genes responsible for the difference in radiation-induced thymocyte apoptosis levels between these two strains are not the same as those responsible for the strain difference in radiation-induced jejunal crypt cell apoptosis levels. The experiments reported here identify strain-specific differences in levels of radiation-induced crypt cell apoptosis and are a first step towards identifying genetic polymorphisms that influence sensitivity of the small intestine to damage from ionizing radiation.

Patients with cancer often experience fatigue and other symptoms even prior to the start of treatment. Because of the strong association between cancer and inflammation it has been hypothesized that these symptoms are a result of tumor-induced inflammation. We sought to test this hypothesis using a syngeneic heterotopic murine model of human papilloma virus-related head and neck cancer. Mice were implanted or not with 1x106 epithelial tonsil cells transfected with E6/7 of HPV16 and HRAS into the hind leg. The resulting tumor expressed high levels of inflammatory cytokines (e.g., IL-6, IL-1beta, and TNF-alpha) and caused elevated cytokine expression within the liver (i.e., IL-6, IL-1beta, and TNF-alpha) and brain (IL-1beta). The tumor reduced burrowing and voluntary wheel running, but did not induce depression-like behavior. To confirm the existence of low grade inflammation, tumor bearing mice were injected with lipopolysaccharide (0.1 mg/kg, IP). Compared to controls, they showed evidence of priming in the form of increased immobility in the tail suspension task and reduced nest building. Chronic treatment with minocycline attenuated tumor-induced burrowing deficits as well as inflammatory cytokine expression without altering tumor response to chemoradation. These data suggest that the tumor-induced inflammation propagates from the tumor to the liver and brain to induce behavioral alterations that may be effectively targeted through the use of anti-inflammatory interventions.