Experiences of the clinical academic pathway: a qualitative study in Greater Manchester to improve the opportunities of minoritised clinical academics.

OBJECTIVES: The aim of this study was to explore the barriers and facilitators faced by clinical academics (CAs) in the Greater Manchester region, with particular attention to the experiences of minoritised groups. DESIGN: A qualitative study using semistructured interviews and focus groups was conducted. A reflexive thematic analysis was applied to identify key themes. SETTING: University of Manchester and National Health Service Trusts in the Greater Manchester region. PARTICIPANTS: The sample of this study was composed of 43 participants, including CAs, senior stakeholders, clinicians and medical and dental students. RESULTS: Six themes were identified. CAs face several barriers and facilitators, some of which-(1) funding insecurity and (2) high workload between the clinic and academia-are common to all the CAs. Other barriers, including (3) discrimination that translates into struggles with self-worth and feeling of not belonging, (4) being or being perceived as foreign and (5) unequal distribution of care duties, particularly affect people from minoritised groups. In contrast, (6) mentorship was commonly identified as one of the most important facilitators. CONCLUSIONS: Cultural and structural interventions are needed, such as introducing financial support for early career CAs and intercalating healthcare students to promote wider social and cultural change and increase the feelings of belonging and representation across the entire CA pipeline.

Radiation enhances the therapeutic effect of Banoxantrone in hypoxic tumour cells with elevated levels of nitric oxide synthase.

Banoxantrone (AQ4N) is a prototype hypoxia selective cytotoxin that is activated by haem containing reductases such as inducible nitric oxide synthase (iNOS). In the present study, we evaluate whether elevated levels of iNOS in human tumour cells will improve their sensitivity to AQ4N. Further, we examine the potential of radiation to increase cellular toxicity of AQ4N under normoxic (aerobic) and hypoxic conditions. We employed an expression vector containing the cDNA for human iNOS to transfect human fibrosarcoma HT1080 tumour cells. Alternatively, parental cells were exposed to a cytokine cocktail to induce iNOS gene expression and enzymatic activity. The cells were then treated with AQ4N alone and in combination with radiation in the presence or absence of the iNOS inhibitor N-methyl-L­arginine. In parental cells, AQ4N showed little difference in toxicity under hypoxic verses normoxic conditions. Notably, cells with upregulated iNOS activity showed a significant increase in sensitivity to AQ4N, but only under conditions of reduced oxygenation. When these cells were exposed to the combination of AQ4N and radiation, there was much greater cell killing than that observed with either modality alone. In the clinical development of hypoxia selective cytotoxins it is likely they will be used in combination with radiotherapy. In the present study, we demonstrated that AQ4N can selectively kill hypoxic cells via an iNOS-dependent mechanism. This hypoxia-selective effect can be augmented by combining AQ4N with radiation without increasing cytotoxicity to well­oxygenated tissues. Collectively, these results suggest that targeting hypoxic tumours with high levels of iNOS with a combination of AQ4N and radiotherapy could be a useful clinical therapeutic strategy.

Glucocorticoid receptor over-expression promotes human small cell lung cancer apoptosis in vivo and thereby slows tumor growth.

Small cell lung cancer (SCLC) is an aggressive tumor, associated with ectopic ACTH syndrome. We have shown that SCLC cells are glucocorticoid receptor (GR) deficient, and that restoration of GR expression confers glucocorticoid sensitivity and induces apoptosis in vitro. To determine the effects of GR expression in vivo, we characterized a mouse SCLC xenograft model that secretes ACTH precursor peptides, and so drives high circulating corticosterone concentrations (analogous to the ectopic ACTH syndrome). Infection of SCLC xenografts with GR-expressing adenovirus significantly slowed tumor growth compared with control virus infection. Time to fourfold initial tumor volume increased from a median of 9 days to 16 days (P=0.05; n=7 per group). Post-mortem analysis of GR-expressing tumors revealed a threefold increase in apoptotic (TUNEL positive) cells (P<0.01). Infection with the GR-expressing adenovirus caused a significant reduction in Bcl-2 and Bcl-xL transcripts. Furthermore, in both the GR-expressing adenovirus-infected cells and tumors, a significant number of uninfected cells underwent apoptosis, supporting a bystander cell killing effect. Therefore, GR expression is pro-apoptotic for human SCLCs in vivo, as well as in vitro, suggesting that loss of GR confers a survival advantage to SCLCs.

The impact of intracellular generation of nitric oxide on the radiation response of human tumor cells.

Nitric oxide (NO) is a potent radiosensitizer of hypoxic mammalian cells. There have been many reports demonstrating radiosensitization in vitro and in vivo by the use of NO donors to generate NO by chemical means or by producing agents that mimic the free radical mechanism(s) of NO for potentiating radiosensitivity. However, much of this work has been done without taking account of the endogenous NO that is generated in tumor cells by NO synthase (NOS) in vitro or in tumor cells and host cells in solid tumors in vivo. To evaluate the contribution of intracellular generated NO to cellular radiosensitivity, we exposed human HT1080 and MDA231 tumor cells to a cytokine cocktail that results in an increase in cellular NOS expression to a level that is seen in many human solid tumors. We also carried out parallel studies to determine the radiosensitivity of HT1080 and MDA231 cells engineered to constitutively overexpress the iNOS gene. When cells are treated with cytokines under anoxic conditions (<0.01% O(2)), there is up to a 9-15-fold increase in NOS expression, but no detectable NO is generated (since O(2) is required for the generation of NO via the NOS-mediated conversion of arginine to citrulline). As a consequence, when these cells are irradiated under hypoxic conditions, no radiosensitization is observed. However, as the oxygen tension was increased, the amount of NO generated also increased, and we show that this NO then contributes to an overall increase in the radiosensitivity of cells. For example, at 1% O(2) in control HT1080 cells, with little measurable NOS activity, the dose of radiation required to reduce survival by 90% was 6 Gy compared to 10 Gy in anoxic conditions. After cytokine treatment, the level of NO generated at 1% O(2) was significantly increased and the dose of radiation needed for 90% cell killing was reduced further to 4 Gy. The presence of the NOS inhibitor N(G)-methyl-l-arginine (NMLA) shortly before and during irradiation ablated this increase in radiosensitivity, confirming that the effect was due to the generation of NO. We conclude that cytokine-mediated up-regulation of the NOS expression in tumor cells can produce sufficient NO to significantly increase the cytotoxic effect of radiation and that this is particularly apparent at intermediate oxygen concentrations.

Effects of cytokine-induced macrophages on the response of tumor cells to banoxantrone (AQ4N).

Tumor-associated macrophages (TAMs) are found in many solid tumors and have often been shown to accumulate in the hypoxic regions surrounding areas of necrosis. TAMs are the major site of expression of nitric oxide synthase (NOS), a heme-containing homodimeric enzyme consisting of oxygenase and reductase domains. The latter has a high degree of sequence homology to cytochrome P450 reductase and a functional consequence of this is the ability of NOS, under hypoxic conditions, to activate the bioreductive drugs tirapazamine and RSU1069. Banoxantrone (AQ4N) is a bioreductive prodrug activated in hypoxia by an oxygen-dependent two-electron reductive process to yield the topoisomerase II inhibitor AQ4. A feature of this process is that the final product could potentially show bystander cell killing. Thus, in this study, we investigated the ability of inducible NOS (iNOS)-expressing TAMs to activate AQ4N and elicit toxicity in cocultured human tumor cells. Murine macrophages were induced to overexpress iNOS by treatment with a combination of cytokines, mixed with HT1080 and HCT116 human tumor cells, and the toxicity of AQ4N was determined under aerobic or hypoxic conditions. The aerobic toxicity of AQ4N toward tumor cells was not affected through coculturing with macrophages. However, under hypoxic conditions, the induction of iNOS activity in the macrophages was associated with an increase in AQ4N metabolism and a substantial increase in tumor cell toxicity, which was dependent on the proportion of macrophages in the culture. This study is the first demonstration of TAM-mediated prodrug activation to result in bystander killing of human tumor cells.

iNOS as a therapeutic target for treatment of human tumors.

Nitric oxide synthase (NOS) has been shown to be overexpressed in a number of human tumors compared to normal tissues and therefore potentially represents an exploitable target in future anticancer therapies. To achieve this, there will be a need to profile tumors to identify those expressing high levels of NOS; alternatively, endogenous (low) levels of NOS could be modulated by induction or through gene therapy approaches. NOS consists of a reductase domain which shares a high degree of sequence homology with P450 reductase and this domain supplies reducing equivalents to a haem containing oxygenase domain that is responsible for the production of nitric oxide. Thus, there are a number of routes of exploitation. Firstly, to take advantage of the reductase domain to activate bioreductive drugs as has been exemplified with tirapazamine and now extended to AQ4N (1,4-bis{2-(dimethylamino-N-oxide)ethylamino}5,8-dihydroxy-anthracene-9,10-dione). Secondly, to take advantage of nitric oxide production for its ability to increase the sensitivity of resistant hypoxic cells to radiation. Lastly, to utilize inhibition of HIF-1 to amplify NO based therapies. In this review we provide examples/evidence of how these objectives can be achieved.

Hypoxia-targeted over-expression of carboxylesterase as a means of increasing tumour sensitivity to irinotecan (CPT-11).

The induced expression of carboxylesterase (CE) enzymes, which convert the prodrug irinotecan (CPT-11) into its active cytotoxic metabolite SN-38, constitutes a promising strategy for cancer gene therapy. By incorporating hypoxia-responsive elements (HREs) in conjunction with the transgene, expression can be targeted specifically to hypoxic tissues (such as solid tumours), expressing the hypoxia-inducible factor 1 (HIF-1). We have constructed a recombinant adenoviral vector, AdHRE-rCE, encoding the cDNA for the highly efficient rabbit liver CE (rCE), under the control of a HRE derived from the human phosphoglycerate kinase 1 (PGK-1) gene in conjunction with a minimal SV40 promoter. In vitro, HT1080 fibrosarcoma and SW480 colon carcinoma cells demonstrated an approximately 10-fold hypoxia-dependent induction in CE expression following pre-infection with AdHRE-rCE, which led to a15-30-fold increased sensitivity to CPT-11. Furthermore, in vivo, SW480 tumour xenografts infected with AdHRE-rCE demonstrated a 2-fold decrease in tumour doubling time, when combined with 7 days of CPT-11 treatment, in comparison to mock-infected controls, with rCE expression shown to be limited to hypoxic regions only. As the cytotoxicity of CPT-11 is reduced under hypoxic conditions, over-expression of a highly efficient CE such as rCE under hypoxia control within these hypoxic cells could reverse this effect and, therefore, form the basis for future clinical treatment strategies.

Reversing hypoxic cell chemoresistance in vitro using genetic and small molecule approaches targeting hypoxia inducible factor-1.

The resistance of hypoxic cells to conventional chemotherapy is well documented. Using both adenovirus-mediated gene delivery and small molecules targeting hypoxia-inducible factor-1 (HIF-1), we evaluated the impact of HIF-1 inhibition on the sensitivity of hypoxic tumor cells to etoposide. The genetic therapy exploited a truncated HIF-1alpha protein that acts as a dominant-negative HIF-1alpha (HIF-1alpha-no-TAD). Its functionality was validated in six human tumor cell lines using HIF-1 reporter assays. An EGFP-fused protein demonstrated that the dominant-negative HIF-1alpha was nucleus-localized and constitutively expressed irrespective of oxygen tension. The small molecules studied were quinocarmycin monocitrate (KW2152), its analog 7-cyanoquinocarcinol (DX-52-1), and topotecan. DX-52-1 and topotecan have been previously established as HIF-1 inhibitors. HT1080 and HCT116 cells were treated with either AdHIF-1alpha-no-TAD or nontoxic concentrations (0.1 microM;

Cancer chemotherapy and drug metabolism.

Drug-metabolizing enzymes and drug transporters are key determinants of the pharmacokinetics and pharmacodynamics of many antineoplastic agents. Metabolism and transport influence the cytotoxic effects of antineoplastic agents in target tumor cells and normal host tissues. This article summarizes several state-of-the-art approaches to enhancing the effectiveness and safety of cancer therapy based on recent developments in our understanding of antineoplastic drug metabolism and transport. Advances in four interrelated research areas presented at a recent symposium sponsored by the Division for Drug Metabolism of the American Society for Pharmacology and Experimental Therapeutics (Experimental Biology 2004; Washington D.C., April 17-21, 2004) are discussed: 1) interactions of anthracyclines with drug-metabolizing enzymes; 2) use of hypoxia-selective gene-directed enzyme prodrug therapy (GDEPT) in combination with bioreductive prodrugs; 3) synergy between glutathione conjugation and conjugate efflux in conferring resistance to electrophilic toxins; and 4) use of cytochromes P450 as prodrug-activating enzymes in GDEPT strategies. A clear theme emerged from this symposium: drug metabolism and transport processes can be modulated and exploited in ways that may offer distinct therapeutic advantages in the management of patients with cancer.

Dual responsive promoters to target therapeutic gene expression to radiation-resistant hypoxic tumor cells.

PURPOSE: Tumor hypoxia is unequivocally linked to poor radiotherapy outcome. This study aimed to identify enhancer sequences that respond maximally to a combination of radiation and hypoxia for use in genetic radiotherapy approaches. METHODS AND MATERIALS: The influence of radiation (5 Gy) and hypoxia (1% O2) on reporter-gene expression driven by hypoxia (HRE) and radiation (Egr-1) responsive elements was evaluated in tumor cells grown as monolayers or multicellular spheroids. Hypoxia-inducible factor-1alpha (HIF-1alpha) and HIF-2alpha protein expression was monitored in parallel. RESULTS: Of the sequences tested, an HRE from the phosphoglycerate kinase-1 gene (PGK-18[5+]) was maximally induced in response to hypoxia plus radiation in all 5 cell lines tested. The additional radiation treatment afforded a significant increase in the induction of PGK-18[5+] compared with hypoxia alone in 3 cell lines. HIF-1alpha/2alpha were induced by radiation but combined hypoxia/radiation treatment did not yield a further increase. The dual responsive nature of HREs was maintained when spheroids were irradiated after delivery of HRE constructs in a replication-deficient adenovirus. CONCLUSIONS: Hypoxia-responsive enhancer element sequences are dually responsive to combined radiation and hypoxic treatment. Their use in genetic radiotherapy in vivo could maximize expression in the most radio-resistant population at the time of radiation and also exploit microenvironmental changes after radiotherapy to yield additional switch-on.

Construction and characterization of multiple human colon cancer cell lines for inducibly regulated gene expression.

Validation of targets for cancer drug discovery requires robust experimental models. Systems based on inducible gene expression are well suited to this purpose but are difficult to establish in several epithelial cell types. Using the recently discovered transcriptional transactivator (rtTA2S-M2), we developed a strategy for fast and efficient generation of Tet On cells. Multiple clones of HCT116, SW480, and HT29 human colon cancer cells for doxycycline-regulated gene expression were constructed that constitutively express green fluorescent protein (GFP) for selection/maintenance purposes. The cell lines displayed good fold inducibility (49-124xHCT116; 178-621xSW480; 261-787xHT29) and minimal leakiness after transient transfection with a luciferase reporter or with vectors driving inducible expression of red fluorescent protein (dsRed2), constitutively active c-Src or dominant negative K-Ras4B. The clones preserved their transformed phenotype as demonstrated by comparing their properties to respective wild type cells, in terms of growth in vitro and in vivo (as tumor xenografts), cell cycle traverse, and sensitivity to drugs used in chemotherapy. These engineered cell lines enabled tightly controlled inducible gene expression both in vitro and in vivo, and proved well suited for construction of double-stable cell lines inducibly expressing a protein of interest. As such they represent a useful research tool for example, to dissect oncogene function(s) in colon cancer. Supplementary material for this article be found at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/94/suppmat_welman.doc.

Hypoxia targeted gene therapy to increase the efficacy of tirapazamine as an adjuvant to radiotherapy: reversing tumor radioresistance and effecting cure.

Solid tumors are characterized by regions of hypoxia that are inherently resistant to both radiotherapy and some chemotherapy. To target this resistant population, bioreductive drugs that are preferentially toxic to tumor cells in a hypoxic environment are being evaluated in clinical trials; the lead compound, tirapazamine (TPZ), is being used in combination with cisplatin and/or with radiotherapy. Crucially, tumor response to TPZ is also dependent on the cellular complement of reductases. In particular, NADPH:cytochrome P450 reductase (P450R) plays a major role in the metabolic activation of TPZ. In a gene-directed enzyme prodrug therapy (GDEPT) approach using adenoviral delivery, we have overexpressed human P450R specifically within hypoxic cells in tumors, with the aim of harnessing hypoxia as a trigger for both enzyme expression and drug metabolism. The adenovirus used incorporates the hypoxia-responsive element (HRE) from the lactate dehydrogenase gene in a minimal SV40 promoter context upstream of the cDNA for P450R. In a human tumor model in which TPZ alone does not potentiate radiotherapeutic outcome (HT1080 fibrosarcoma), we witnessed complete tumor regression when tumors were virally transduced before treatment.

Viral delivery of P450 reductase recapitulates the ability of constitutive overexpression of reductase enzymes to potentiate the activity of mitomycin C in human breast cancer xenografts.

Indolequinones such as mitomycin C (MMC) require enzymatic bioreduction to yield cytotoxic moieties. An attractive approach to overcome the potential variability in reductive bioactivation between tumors is to exploit specific enzyme-bioreductive drug combinations in an enzyme-directed gene therapy (GDEPT) approach. To this end, human breast cancer cell lines (T47D, MDA468, and MDA231) that overexpress either DT-diaphorase (DTD) or NADPH:cytochrome P450 reductase (P450R) have been developed. Cytotoxicity of MMC was evaluated in the panel of cell lines following aerobic or anoxic exposure in vitro. DTD and/or P450R overexpression sensitized cells to MMC in air with no further increase in the cytotoxicity of MMC under anoxia. The most profound effect was seen in the MDA468 cells, where a 27-fold increase in potency was observed for MMC in the DTD-overexpressing cell line. The MMC sensitization achieved through DTD and P450R overexpression in MDA468 cells was maintained in vivo. Xenografts established from the clonal lines exhibited significant tumor control following MMC treatment (treated/control [T/C] 17% and 51% for DTD and P450R xenografts, respectively) that was not seen in wild-type tumors (T/C 102%). Delivery of a clinically relevant adenoviral vector encoding P450R to MDA468 wild-type tumors yielded comparable P450R activity to that seen in the P450R clonal xenografts and resulted in greater MMC sensitization (T/C 46%). The model systems developed will facilitate the identification of novel indolequinone agents that are targeted toward a specific enzyme for bioactivation and are consequently of potential use in a GDEPT approach.

Non-nuclear localized human NOSII enhances the bioactivation and toxicity of tirapazamine (SR4233) in vitro.

Tirapazamine (TPZ) is the lead member of a class of bioreductive drugs currently in phase II and III clinical trials. TPZ requires metabolic activation to give a cytotoxic free radical species, and this hypoxia-mediated process is carried out by a variety of cellular reductases, including NADPH cytochrome c (P450) reductase (P540R). Nitric-oxide synthase (NOS) is widely expressed in human tumors, and this enzyme consists of an oxidase and a reductase domain, the latter showing striking homology to P450R. Thus, in this article, we have investigated the role of one of the cytosolic isoforms of NOS [inducible NOS (NOSII)] in the bioactivation of this DNA-damaging antitumor agent. To achieve this, we have constitutively overexpressed NOSII in human breast tumor MDA231 cells by employing an optimized expression vector in which the strong human polypeptide chain elongation factor 1alpha promoter drives a bicistronic message encoding the genes for human NOSII and the puromycin-resistant gene (pac). Subcellular localization of NOSII in the stably transfected clones was determined after differential centrifugation and showed that NOSII catalytic activity was exclusively cytosolic as determined by conventional activity assay. This was confirmed by immunostaining followed by fluorescent microscopy studies. The increase in NOSII activity in a series of transfected clones was associated with an increase in TPZ metabolism and toxicity under hypoxic conditions. There was no similar increase in aerobic toxicity. These findings are of significance for two reasons. First, cellular NOSII activity, similar to that seen in human breast cancer, could contribute to TPZ toxicity; second, this will be a result of NOS-derived/cytosol-associated TPZ radicals.

Combining bioreductive drugs and radiation for the treatment of solid tumors.

Methods now exist for the identification of human tumors that contain significant numbers of hypoxic cells and are thereby suitable for treatment with bioreductive drugs to eliminate this refractory cell population. However, to fully exploit the potential of bioreductive drugs, they will need to be used in combination with other modalities likely to target the proliferating aerobic cells in the tumor. Radiation is the treatment that is most effective in killing aerobic cells; therefore, the present report reviews the available preclinical data on combined radiation/bioreductive drug treatments.

Adenovirus vector-mediated delivery of the prodrug-converting enzyme carboxypeptidase G2 in a secreted or GPI-anchored form: High-level expression of this active conditional cytotoxic enzyme at the plasma membrane.

Carboxypeptidase G2 (CPG2) is a powerful prodrug-converting enzyme. Without a requirement for endogenous enzymes or cofactors, it can directly activate mustard alkylating prodrugs to cytotoxic species, killing both quiescent and dividing cells. This paper provides the first report of its use in the context of a clinically relevant delivery vehicle using adenovirus vectors. To strengthen the efficacy of the prodrug-activating system, the enzyme has been engineered to be secreted or glycosylphosphatidylinositol (GPI) anchored to the extracellular membrane of tumor cells, resulting in an enhanced bystander effect by facilitating diffusion of the active drug through extracellular, rather than intracellular, activation. Using the vectors, we have achieved expression of functional secreted or GPI-anchored CPG2 in a panel of tumor cell lines demonstrating no loss in efficacy as a result of GPI anchor retention. Despite variable transduction efficiencies inherent to these vectors, greater than 50% cell kill was achievable in all of the cell lines tested following only a single exposure to the prodrug ZD2767P. Even in cell lines refractive to infection with the vectors, substantial cell death was recorded, indicative of the enhanced bystander effect generated following extracellular prodrug activation. A direct evaluation of the efficacy of our system has been made against adenoviral delivery of herpes simples virus thymidine kinase plus ganciclovir (GCV), a suicide gene therapy approach already in the clinic. In a short-term human glioma culture (IN1760) resistant to the clinical chemotherapeutic drug CCNU (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea), thymidine kinase/GCV effected no cell killing compared to 70% cell killing with our system.

Antiangiogenic, bioreductive and gene therapy approaches to the treatment of hypoxic tumours.

Quinone based bioreductive drugs have, potentially, a very versatile use in cancer chemotherapy. They can be activated by DT-diaphorase and hence can be used to target tumour types rich in this (O2)-independent reductase enzyme. Small molecular modifications can substantially reduce specificity for DT-diaphorase and under these circumstances the quinones become much less toxic in air but retain their potent cytotoxic effects under hypoxic conditions. Our understanding of the reductive (bio) chemistry of indolequinones, in particular, has subsequently allowed us to develop a platform technology where almost any therapeutic entity can potentially be delivered, selectively, to hypoxic tumours. Antiangiogenic approaches are currently receiving a substantial amount of attention and this review brings their development into context in view of the hypoxia dependence for neovascularization. Lastly, the use of bioreductive drugs when combined with hypoxia-mediated gene therapy is described. Such an approach provides a unique dual level of specificity for targeting hypoxic tumours and potentially can provide substantial therapeutic benefit.