Potential for a novel manganese porphyrin compound as adjuvant canine lymphoma therapy.

PURPOSE: Manganese porphyrins are redox-active drugs and superoxide dismutase mimics, which have been shown to chemosensitize lymphoma, a cancer which frequently occurs in dogs. This study aimed to identify critical information regarding the pharmacokinetics and toxicity of Mn(III) meso-tetrakis (N-n-butoxyetylpyridium-2-yl) porphyrin, (MnTnBuOE-2-PyP5+, MnBuOE) in dogs as a prelude to a clinical trial in canine lymphoma patients. METHODS: A single-dose pharmacokinetic (PK) study in normal dogs was performed to determine the plasma half-life (t 1/2) of MnBuOE. A dose reduction study was performed to establish the maximum tolerated dose (MTD) of MnBuOE. The safety and PK of a multi-dosing protocol was assessed. RESULTS: Peak plasma drug concentration occurred 30 min post-injection. The t 1/2 was defined as 7 h. MnBuOE induced an anaphylactic reaction and prolonged tachycardia. The MTD was defined as 0.25 mg/kg. The dogs were given MTD 3×/week for 2-3 weeks. The highest recorded tissue drug levels were in the lymph nodes (4-6 µM), followed by kidney and liver (2.5, 2.0 uM, respectively). CONCLUSIONS: We obtained critical information regarding the PK and toxicity of MnBuOE in dogs. The acute drug reaction and tachycardia post-injection have not been described in other species and may be specific to canines. The high tissue drug levels in lymph nodes have not been previously reported. MnBuOE accumulation in lymph nodes has important implications for the utility of adjuvant MnBuOE to treat lymphoma. With MnBuOE lymph node accumulation, reduction in the dose and/or administration frequency could be possible, leading to reduced toxicity.

Akt phosphorylates and activates HSF-1 independent of heat shock, leading to Slug overexpression and epithelial-mesenchymal transition (EMT) of HER2-overexpressing breast cancer cells.

Epithelial-mesenchymal transition (EMT) is an essential step for tumor progression, although the mechanisms driving EMT are still not fully understood. In an effort to investigate these mechanisms, we observed that heregulin (HRG)-mediated activation of HER2, or HER2 overexpression, resulted in EMT, which is accompanied with increased expression of a known EMT regulator Slug, but not TWIST or Snail. We then investigated how HER2 induced Slug expression and found, for the first time, that there are four consensus HSF sequence-binding elements (HSEs), the binding sites for heat shock factor-1 (HSF-1), located in the Slug promoter. HSF-1 bound to and transactivated the Slug promoter independent of heat shock, leading to Slug expression in breast cancer cells. Mutation of the putative HSEs ablated Slug transcriptional activation induced by HRG or HSF-1 overexpression. Knockdown of HSF-1 expression by siRNA reduced Slug expression and HRG-induced EMT. The positive association between HSF-1 and Slug was confirmed by immunohistochemical staining of a cohort of 100 invasive breast carcinoma specimens. While investigating how HER2 activated HSF-1 independent of heat shock, we observed that HER2 activation resulted in concurrent phosphorylation of Akt and HSF-1. We then observed, also for the first time, that Akt directly interacted with HSF-1 and phosphorylated HSF-1 at S326. Inhibition of Akt using siRNA, dominant-negative Akt mutant, or small molecule inhibitors prevented HRG-induced HSF-1 activation and Slug expression. Conversely, constitutively active Akt induced HSF-1 phosphorylation and Slug expression. HSF-1 knockdown reduced the ability of Akt to induce Slug expression, indicating an essential role that HSF-1 plays in Akt-induced Slug upregulation. Altogether, our study uncovered the existence of a novel Akt-HSF-1 signaling axis that leads to Slug upregulation and EMT, and potentially contributes to progression of HER2-positive breast cancer.

Carbonic anhydrase IX is a predictive marker of doxorubicin resistance in early-stage breast cancer independent of HER2 and TOP2A amplification.

BACKGROUND: In early-stage breast cancer, adjuvant chemotherapy is associated with significant systemic toxicity with only a modest survival benefit. Therefore, there is considerable interest in identifying predictive markers of response to therapy. Doxorubicin, one of the most common drugs used to treat breast cancer, is an anthracycline chemotherapeutic agent, a class of drugs known to be affected by hypoxia. Accordingly, we examined whether expression of the endogenous hypoxia marker carbonic anhydrase IX (CA IX) is predictive of outcome in early-stage breast cancer patients treated with doxorubicin. METHODS: We obtained 209 early-stage pre-treatment surgically-resected breast tumours from patients, who received doxorubicin in their chemotherapeutic regimen and had >10 years of follow-up. Immunohistochemistry was used to detect CA IX, and we used fluorescence in situ hybridisation to detect both human epidermal growth factor receptor (HER2) and DNA topoisomerase II-alpha (TOP2A) gene amplification. RESULTS: Carbonic anhydrase IX intensity was significantly correlated with progression-free survival (PFS) and overall survival (OS) in patients receiving 300 mg m(-2) of doxorubicin (HR=1.82 and 3.77; P=0.0014 and 0.010, respectively). There was a significant, inverse correlation between CA IX score and oestrogen receptor expression, but no significant correlations were seen with either HER2 or TOP2A ratio. CONCLUSION: We demonstrate that CA IX expression is correlated with worse PFS and OS for breast cancer patients treated with doxorubicin, independent of HER2 or TOP2A gene amplification. This study provides evidence that using CA IX to detect hypoxia in surgically-resected breast tumours may be of clinical use in choosing an appropriate chemotherapy regimen.

SU-E-I-07: Proposal for a Novel Algorithm of Effective Dose Evaluation for Medical Radiation Exposure.

PURPOSE: The validity of effective dose (ED) in medical applications, which mostly involving partial-organ irradiation, has long been argued. We now propose a new algorithm of effective dose evaluation for medical radiation exposure. The new algorithm will include new Relative Volume Factors, or RVFs, to account for the relative irradiated volume of the partially-irradiated organs, such as skin and bone marrow. METHODS: To validate the proposed algorithm, three ED evaluation methods were applied to five neuro-imaging protocols of a C-arm cone-beam CT: 1) MOSFET-anthropomorphic- phantom measurement for organ doses, ED evaluated with the original ICRP algorithm; 2) using the same MOSFET-measured organ doses, ED evaluated with the new algorithm; and 3) Monte Carlo (PCXMC, developed by STUK) dose estimation as gold standard. RESULTS: For all five imaging protocols, the ED evaluations by the new algorithm agreed very well with the Monte Carlo simulation results (within 10% difference), whereas the original ICRP algorithm overestimated EDs by approximately a factor of 2. CONCLUSIONS: The proposed algorithm provides a more accurate estimation of ED in medical exposures than the original ICRP algorithm. It potentially establishes the ED as a more reasonable index for cross-protocol dose comparison.

Upregulation of VEGF-A and CD24 gene expression by the tGLI1 transcription factor contributes to the aggressive behavior of breast cancer cells.

The Hedgehog signaling pathway is one of the most dysregulated pathways in human cancers. The glioma-associated oncogene homolog 1 (GLI1) transcription factor is the terminal effector of the Hedgehog pathway, frequently activated in human breast cancer and an emerging target of breast cancer therapy. While somatic mutations in the human GLI1 gene have never been reported in any cell or tumor type, we recently uncovered the existence of a novel alternatively spliced, truncated GLI1 (tGLI1) that has an in-frame deletion of 41 codons spanning the entire exon 3 and part of exon 4 of the GLI1 gene. Using glioblastoma models, we showed that tGLI1 has gained the ability to promote glioblastoma migration and invasion via its gain-of-function transcriptional activity. However, the pathological impact of tGLI1 on breast cancer remains undefined. Here, we report that tGLI1 is frequently expressed in human breast cancer cell lines and primary specimens we have examined to date, but is undetectable in normal breast tissues. We found for the first time that tGLI1, but not GLI1, binds to and enhances the human vascular endothelial growth factor-A (VEGF-A) gene promoter, leading to its upregulation. Consequently, tGLI1-expressing MDA-MB-231 breast cancer cells secret higher levels of VEGF-A and contain a higher propensity, than the isogenic cells with control vector and GLI1, to stimulate in vitro angiogenesis of human vascular endothelial cells. We further showed that tGLI1 has gained the ability to enhance the motility and invasiveness of breast cancer cells in a proliferation-independent manner and that this functional gain is associated with increased expression of migration/invasion-associated genes, CD24, MMP-2 and MMP-9. tGLI1 has also acquired the property to facilitate anchorage-independent growth of breast cancer cells. Collectively, our results define tGLI1 as a gain-of-function GLI1 transcription factor and a novel mediator of the behavior of clinically more aggressive breast cancer.

ß-arrestin1 mediates metastatic growth of breast cancer cells by facilitating HIF-1-dependent VEGF expression.

ß-Arrestins 1 and 2 are multifunctional adaptor proteins originally discovered for their role in desensitizing seven-transmembrane receptor signaling via the heterotrimeric guanine nucleotide-binding proteins. Recently identified roles of ß-arrestins include regulation of cancer cell chemotaxis and proliferation. Herein, we report that ß-arrestin1 expression regulates breast tumor colonization in nude mice and cancer cell viability during hypoxia. ß-Arrestin1 robustly interacts with nuclear hypoxia-induced factor-1α (HIF-1α) that is stabilized during hypoxia and potentiates HIF-1-dependent transcription of the angiogenic factor vascular endothelial growth factor-A (VEGF-A). Increased expression of ß-arrestin1 in human breast cancer (infiltrating ductal carcinoma or IDC and metastatic IDC) correlates with increased levels of VEGF-A. While the anti-angiogenic drug thalidomide inhibits HIF-1-dependent VEGF transcription in breast carcinoma cells, it does not prevent HIF-1α stabilization, but leads to aberrant localization of HIF-1α to the perinuclear compartments and surprisingly stimulates nuclear export of ß-arrestin1. Additionally, imatinib mesylate that inhibits release of VEGF induces nuclear export of ß-arrestin1-HIF-1α complexes. Our findings suggest that ß-arrestin1 regulates nuclear signaling during hypoxia to promote survival of breast cancer cells via VEGF signaling and that drugs that induce its translocation from the nucleus to the cytoplasm could be useful in anti-angiogenic and breast cancer therapies.

A novel angiopoietin-derived peptide displays anti-angiogenic activity and inhibits tumour-induced and retinal neovascularization.

BACKGROUND AND PURPOSE: Pathological angiogenesis is associated with various human diseases, such as cancer, autoimmune diseases and retinopathy. The angiopoietin (Ang)-Tie2 system plays critical roles in several steps of angiogenic remodelling. Here, we have investigated the anti-angiogenic effect of a novel angiopoietin-derived peptide. EXPERIMENTAL APPROACH: Using computational methods, we identified peptides from helical segments within angiopoietins, which were predicted to inhibit their activity. These peptides were tested using biochemical methods and models of angiogenesis. The peptide with best efficacy, A11, was selected for further characterization as an anti-angiogenic compound. KEY RESULTS: The potent anti-angiogenic activity of A11 was demonstrated in a multicellular assay of angiogenesis and in the chorioallantoic membrane model. A11 bound to angiopoietins and reduced the binding of Ang-2 to Tie2. A11 was also significantly reduced vascular density in a model of tumour-induced angiogenesis. Its ability to inhibit Ang-2 but not Ang-1-induced endothelial cell migration, and to down-regulate Tie2 levels in tumour microvessels, suggests that A11 targets the Ang-Tie2 pathway. In a rat model of oxygen-induced retinopathy, A11 strongly inhibited retinal angiogenesis. Moreover, combination of A11 with an anti-VEGF antibody showed a trend for further inhibition of angiogenesis, suggesting an additive effect. CONCLUSIONS AND IMPLICATIONS: Our results indicate that A11 is a potent anti-angiogenic compound, through modulation of the Ang-Tie2 system, underlining its potential as a therapeutic agent for the treatment of ocular and tumour neovascularization, as well as other pathological conditions that are dependent on angiogenesis.

Improving the quantitative accuracy of optical-emission computed tomography by incorporating an attenuation correction: application to HIF1 imaging.

Optical computed tomography (optical-CT) and optical-emission computed tomography (optical-ECT) are new techniques for imaging the 3D structure and function (including gene expression) of whole unsectioned tissue samples. This work presents a method of improving the quantitative accuracy of optical-ECT by correcting for the 'self'-attenuation of photons emitted within the sample. The correction is analogous to a method commonly applied in single-photon-emission computed tomography reconstruction. The performance of the correction method was investigated by application to a transparent cylindrical gelatin phantom, containing a known distribution of attenuation (a central ink-doped gelatine core) and a known distribution of fluorescing fibres. Attenuation corrected and uncorrected optical-ECT images were reconstructed on the phantom to enable an evaluation of the effectiveness of the correction. Significant attenuation artefacts were observed in the uncorrected images where the central fibre appeared approximately 24% less intense due to greater attenuation from the surrounding ink-doped gelatin. This artefact was almost completely removed in the attenuation-corrected image, where the central fibre was within approximately 4% of the others. The successful phantom test enabled application of attenuation correction to optical-ECT images of an unsectioned human breast xenograft tumour grown subcutaneously on the hind leg of a nude mouse. This tumour cell line had been genetically labelled (pre-implantation) with fluorescent reporter genes such that all viable tumour cells expressed constitutive red fluorescent protein and hypoxia-inducible factor 1 transcription-produced green fluorescent protein. In addition to the fluorescent reporter labelling of gene expression, the tumour microvasculature was labelled by a light-absorbing vasculature contrast agent delivered in vivo by tail-vein injection. Optical-CT transmission images yielded high-resolution 3D images of the absorbing contrast agent, and revealed highly inhomogeneous vasculature perfusion within the tumour. Optical-ECT emission images yielded high-resolution 3D images of the fluorescent protein distribution in the tumour. Attenuation-uncorrected optical-ECT images showed clear loss of signal in regions of high attenuation, including regions of high perfusion, where attenuation is increased by increased vascular ink stain. Application of attenuation correction showed significant changes in an apparent expression of fluorescent proteins, confirming the importance of the attenuation correction. In conclusion, this work presents the first development and application of an attenuation correction for optical-ECT imaging. The results suggest that successful attenuation correction for optical-ECT is feasible and is essential for quantitatively accurate optical-ECT imaging.

Use of bioluminescence imaging to detect enhanced hepatic and systemic tumor growth following partial hepatectomy in mice.

BACKGROUND: The impact of partial hepatectomy on intra-hepatic and distant tumor growth is a matter of controversy. Utilizing a highly sensitive tumor imaging strategy, we sought to demonstrate whether this growth-acceleration occurs, and to develop an animal model with which to investigate potential therapeutic strategies. METHODS: Mice bearing constitutively-active luciferase-expressing tumor cells were subjected to either 70% partial hepatectomy (PH; n=10) or a sham operation (n=11). Mice were sacrificed 14 days later and remnant livers (or anatomic equivalents in the control group) and lungs harvested for bioluminescence detection. RESULTS: Remnant liver weights were significantly increased in PH compared to equivalent lobes in sham-operated animals (t-test; p=0.005). Tumor burden as measured by bioluminescence was significantly higher in both liver and lung specimens in the PH group (Wilcoxon's Rank Sum test; p=0.01 and 0.004, respectively). CONCLUSIONS: Following PH, enhanced metastatic growth was depicted regionally and systemically with bioluminescence imaging providing an objective measure of tumor burden. This preclinical model can help to identify adjuvant therapies that can influence both tumor growth and liver regeneration.

The role of hypoxia in canine cancer.

Human oncology has clearly demonstrated the existence of hypoxic tumours and the problematic nature of those tumours. Hypoxia is a significant problem in the treatment of all types of solid tumours and a common reason for treatment failure. Hypoxia is a negative prognostic indicator of survival and is correlated with the development of metastatic disease. Resistance to radiation therapy and chemotherapy can be because of hypoxia. There are two dominant types of hypoxia recognized in tumours, static and intermittent. Both types of hypoxia are important in terms of resistance. A variety of physiological factors cause hypoxia, and in turn, hypoxia can induce genetic and physiological changes. A limited number of studies have documented that hypoxia exists in spontaneous canine tumours. The knowledge from the human literature of problematic nature of hypoxic tumours combined with the rapid growth of veterinary oncology has necessitated a better understanding of hypoxia in canine tumours.

Treatment with imatinib improves drug delivery and efficacy in NSCLC xenografts.

Imatinib, an inhibitor of PDGF-Rbeta and other tyrosine kinase receptors, has been shown to decrease microvessel density and interstitial fluid pressure in solid tumours, thereby improving subsequent delivery of small molecules. The purpose of this study was to test whether pretreatment with imatinib increases the efficacy of traditional chemotherapy in mice bearing non-small cell lung carcinoma xenografts, and to investigate the effects of imatinib on liposomal drug delivery. Efficacy treatment groups included (n=9-10): saline control, imatinib alone (oral gavage, 100 mg kg(-1) x 7 days), docetaxel alone (10 mg kg(-1) i.p. 2 x /week until killing), and imatinib plus docetaxel (started on day 7 of imatinib). Tumours were monitored until they reached four times the initial treatment volume (4 x V) or 28 days. A separate experiment compared tumour doxorubicin concentrations (using high performance liquid chromatography) 24 h after treatment with liposomal doxorubicin alone (6 mg kg(-1) i.v., n=9) or imatinib plus liposomal doxorubicin (n=16). Imatinib plus docetaxel resulted in significantly improved antitumour efficacy (0/10 animals reached 4 x V by 28 days) when compared to docetaxel alone (3/9 reached 4 x V, P=0.014) or imatinib alone (9/10 reached 4 x V, P=0.025). Pretreatment with imatinib also significantly increased tumour concentrations of liposomal doxorubicin. Overall, these preclinical studies emphasise the potential of imatinib as an adjunct to small molecule or liposomal chemotherapy.

Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during optical clearing.

Optical emission computed tomography (optical-ECT) is a technique for imaging the three-dimensional (3D) distribution of fluorescent probes in biological tissue specimens with high contrast and spatial resolution. In optical-ECT, functional information can be imaged by (i) systemic application of functional labels (e.g. fluorophore labelled proteins) and/or (ii) endogenous expression of fluorescent reporter proteins (e.g. red fluorescent protein (RFP), green fluorescent protein (GFP)) in vivo. An essential prerequisite for optical-ECT is optical clearing, a procedure where tissue specimens are made transparent to light by sequential perfusion with fixing, dehydrating and clearing agents. In this study, we investigate clearing protocols involving a selection of common fixing (4% buffered paraformaldehyde (PFA), methanol and ethanol), dehydrating (methanol and ethanol) and clearing agents (methyl salicylate and benzyl-alcohol-benzyl-benzoate (BABB)) in order to determine a 'fluorescence friendly' clearing procedure. Cell culture experiments were employed to optimize the sequence of chemical treatments that best preserve fluorescence. Texas red (TxRed), fluorescein isothiocyanate (FITC), RFP and GFP were tested as fluorophores and fluorescent reporter proteins of interest. Fluorescent and control cells were imaged on a microscope using a DSred2 and FITC filter set. The most promising clearing protocols of cell culture experiments were applied to whole xenograft tumour specimens, to test their effectiveness in large unsectioned samples. Fluorescence of TxRed/FITC fluorophores was not found to be significantly affected by any of the test clearing protocols. RFP and GFP fluorescence, however, was found to be significantly greater when cell fixation was in ethanol. Fixation in either PFA or methanol resulted in diminished fluorescence. After ethanol fixation, the RFP and GFP fluorescence proved remarkably robust to subsequent exposure to either methyl salicylate or BABB. The optimized optical clearing procedure of ethanol fixation followed by methyl salicylate clearing preserved the fluorescence of constitutive RFP in whole xenograft tumour specimens, about 1 cc in dimension, indicating successful extension from cell plating experiments to whole tissue samples. Finally, the feasibility of imaging the 3D distribution of viable tumour cells (as indicated by the RFP emission) is demonstrated by optical-ECT imaging of cleared xenograft tumours using an in-house system.

Treatment with Imatinib in NSCLC is associated with decrease of phosphorylated PDGFR-beta and VEGF expression, decrease in interstitial fluid pressure and improvement of oxygenation.

Elevated intratumoral interstitial fluid pressure (IFP) and tumour hypoxia are independent predictive factors for poor survival and poor treatment response in cancer patients. However, the relationship between IFP and tumour hypoxia has not yet been clearly established. Preclinical studies have shown that lowering IFP improves treatment response to cytotoxic therapy. Interstitial fluid pressure can be reduced by inhibition of phosphorylated platelet-derived growth factor receptor-beta (p-PDGFR-beta), a tyrosine kinase receptor frequently overexpressed in cancer stroma, and/or by inhibition of VEGF, a growth factor commonly overexpressed in tumours overexpressing p-PDGFR-beta. We hypothesised that Imatinib, a specific PDGFR-beta inhibitor will, in addition to p-PDGFR-beta inhibition, downregulate VEGF, decrease IFP and improve tumour oxygenation. A549 human lung adenocarcinoma xenografts overexpressing PDGFR-beta were grown in nude mice. Tumour-bearing animals were randomised to control and treatment groups (Imatinib 50 mg kg(-1) via gavage for 4 days). Interstitial fluid pressure was measured in both groups before and after treatment. EF5, a hypoxia marker, was administered 3 h before being killed. Tumours were sectioned and stained for p-PDGFR-beta, VEGF and EF5 binding. Stained sections were viewed with a fluorescence microscope and image analysis was performed. Imatinib treatment resulted in significant reduction of p-PDGFR-beta, VEGF and IFP. Tumour oxygenation was also significantly improved. This study shows that p-PDGFR-beta-overexpressing tumours can be effectively treated with Imatinib to decrease tumour IFP. Importantly, this is the first study demonstrating that Imatinib treatment improves tumour oxygenation and downregulates tumour VEGF expression.

Changes in tumour oxygenation during fractionated hyperthermia and radiation therapy in spontaneous canine sarcomas.

Tumour oxygenation was measured in seven canine soft tissue sarcomas being treated with a fractionated course of radiation and hyperthermia. Measurements obtained during treatment were compared to pre-treatment measurements. The most important finding was an increase in oxygenation in tumours with low pre-treatment oxygenation that persisted throughout treatment. This is an advantageous hyperthermia effect as it may lead to increased radiation cell killing at each fraction. In other tumours, potentially less advantageous changes in oxygenation may be hyperthermia fractionation related and this deserves further investigation.

ROS production and angiogenic regulation by macrophages in response to heat therapy.

PURPOSE: It has been well established that inadequate blood supply combined with high metabolic rates of oxygen consumption results in areas of low oxygen tension (<1%) within malignant tumours and that elevating tumour temperatures above 39 degrees Celsius results in significant improvement in tumour oxygenation. Macrophages play a dual role in tumour initiation and progression having both pro-tumour and anti-tumour effects. However, the response of macrophages to heat within a hypoxic environment has not yet been clearly defined. METHODS: Raw 264.7 murine macrophages were incubated under normoxia and chronic hypoxia at temperatures ranging from 37-43 degrees Celsius. Under normoxia at 41 degrees Celsius, macrophages start to release significant levels of superoxide. The combination of heat with hypoxia constitutes an additional stimulus leading to increased respiratory burst of macrophages. RESULTS: The high levels of superoxide were found to be associated with changes in macrophage production of pro-angiogenic cytokines. While hypoxia alone (37 degrees Celsius) increased levels of hypoxia inducible factor-1alpha (HIF-1alpha) in macrophages, the combination of hypoxia and mild hyperthermia (39-41 degrees Celsius) induced a strong reduction in HIF-1alpha expression. The HIF-regulated vascular endothelial growth factor (VEGF) decreased simultaneously, revealing that heat inhibits both HIF-1alpha stabilization and transcriptional activity. CONCLUSION: The data suggest that temperatures which are readily achievable in the clinic (39-41 degrees Celsius) might be optimal for maximizing hyperthermic response. At higher temperatures, these effects are reversed, thereby limiting the therapeutic benefits of more severe hyperthermic exposure.

HIF-1 and tumour radiosensitivity.

Hypoxia-inducible factor-1 (HIF-1) plays important roles in regulating radiosensitivity, making it a potentially promising target for tumour radiosensitisation. Here, we discuss the rationale for, and the potential pitfalls of, combining HIF-1 blockade with radiotherapy. In doing so, we describe clinical scenarios in which HIF-1 inhibition might optimise tumour radiosensitivity.

Apoptotic DNA fragmentation factor maintains chromosome stability in a P53-independent manner.

DNA fragmentation factor (DFF)/caspase-activated DNase (CAD) is responsible for DNA fragmentation, a hallmark event during apoptosis. Although DNA fragmentation is an evolutionarily conserved process across species, its biological function is not clearly understood. In this study, we constructed cell lines expressing a mutant ICAD (inhibitor of CAD) protein that is resistant to caspase cleavage and therefore constantly binds to DFF/CAD and inhibits DNA fragmentation. We found that irradiation of these cells led to increased chromosome aberrations and aneuploidy when compared with their parental controls. The increased chromosome instability is observed irrespective of cellular P53 status, suggesting that the effect of DFF/CAD is independent of P53. Inhibition of apoptotic DNA fragmentation resulted in increased clonogenic survival of irradiated cells and a delay in removal of cells with DNA damages induced by radiation, an effect similar to that in cells with p53 mutations. Consistent with DFF/CAD's effect on clonogenic survival, tumors established from cells deficient in DNA fragmentation showed enhanced growth in nude mice. Therefore, our results suggest that DFF/CAD plays an important and P53-independent role in maintaining chromosome stability and suppressing tumor development.

Human recombinant erythropoietin (rEpo) has no effect on tumour growth or angiogenesis.

Tumour hypoxia has been shown to increase mutation rate, angiogenesis, and metastatic potential, and decrease response to conventional therapeutics. Improved tumour oxygenation should translate into increased treatment response. Exogenous recombinant erythropoietin (rEpo) has been recently shown to increase tumour oxygenation in a mammary carcinoma model. The mechanism of this action is not yet understood completely. The presence of Epo and its receptor (EpoR) have been demonstrated on several normal and neoplastic tissues, including blood vessels and various solid tumours. In addition, rEpo has been shown in two recent prospective, randomized clinical trials to negatively impact treatment outcome. In this study, we attempt to characterize the direct effects of rEpo on tumour growth and angiogenesis in two separate rodent carcinomas. The effect of rEpo on R3230 rat mammary adenocarcinomas, CT-26 mouse colon carcinomas, HCT-116 human colon carcinomas, and FaDu human head and neck tumours, all of which express EpoR, was examined. There were no differences in tumour growth or proliferation (measured by Ki-67) between placebo-treated and rEpo-treated tumours. In the mammary window chamber, vascular length density (VLD) measurements in serial images of both placebo-treated and Epo-treated rats revealed no difference in angiogenesis between the Epo-treated tumours and placebo-treated tumours at any time point. These experiments are important because they suggest that the recent clinical detriment seen with the use of Epo is not due to its tumour growth effects or angiogenesis. These studies also suggest that further preclinical studies need to examine rEpo's direct tumour effects in efforts to improve the therapeutic benefits of Epo in solid tumour patients.

Phase I/II trial of intravenous Doxil and whole abdomen hyperthermia in patients with refractory ovarian cancer.

OBJECTIVE: A phase I/II study of Doxil combined with whole abdomen hyperthermia was conducted in patients with refractory ovarian cancer. Liposomal doxorubicin combined with hyperthermia has been shown to increase both liposomal delivery and drug extravasation into tumour xenografts resulting in enhanced cytotoxic effects. PATIENTS AND METHODS: Thirty patients with either recurrent or persistent epithelial ovarian cancer were enrolled. All patients had either measurable or assessable disease. Patients received intravenous (IV) Doxil at a dose of 40 mg m-2 as a 1-h infusion followed by whole abdomen hyperthermia. The phase I portion of the study was performed to determine the maximal tolerated dose (MTD) of hyperthermia. Quality of life (QoL) was performed at baseline, prior to each cycle and every 3 months. Plasma pharmacokinetic studies were performed with the first cycle. RESULTS: Ten patients participated in the phase I portion of the study which demonstrated that the MTD of hyperthermia was 60 min after either average vaginal and rectal temperatures of 40 degrees C had been achieved or after 30 min of power application, whichever was shorter. All 30 patients were either paclitaxel and/or platinum resistant initially or developed resistant disease. The median number of prior chemotherapeutic regimens was three (range 2-8) and six patients had been previously treated with Doxil. There were three partial responses for a response rate of 10% (95% CI: [2%, 27%]) and eight patients (27%; 95% CI: [12%, 46%]) had disease stabilization. The median time to progression or death was 3.4 months (95% CI: [2.6, 5.2]) and the median survival was 10.8 months (95% CI: [8.8, 17.4]). Twelve patients (40%) experienced palmar-plantar erythrodysesthesia (PPE), but only four (13%) experienced grade 3-4 PPE toxicity. Doxil systemic exposure was higher in those with grade 3-4 PPE compared to those with no PPE. None of the patients had grade 3-4 thermal toxicity due to hyperthermia. QoL was not decreased in patients responding to therapy. CONCLUSIONS: Therapy with intravenous Doxil and whole abdomen hyperthermia for patients with platinum/paclitaxel resistant ovarian cancer is feasible and does not negatively impact quality of life.