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The colony formation assay is the gold-standard technique to assess cell viability after treatment with cytotoxic reagents, ionizing radiation, and cytotoxic combinatorial treatments. This protocol describes a high-throughput automated and high-content imaging approach to screen siRNA molecular libraries in HeLa cervical cancer cells in 96-well format. We detail reverse transfection of cells with siRNAs, followed by ionizing radiation, fixing, and staining of the plates for automated colony counting. This protocol can be used across a broad range of cell types. For complete details on the use and execution of this protocol, please refer to Tiwana et al. (2015)., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.

Purpose: Tumor hypoxia fuels an aggressive tumor phenotype and confers resistance to anticancer treatments. We conducted a clinical trial to determine whether the antimalarial drug atovaquone, a known mitochondrial inhibitor, reduces hypoxia in non-small cell lung cancer (NSCLC)., Patients and Methods: Patients with NSCLC scheduled for surgery were recruited sequentially into two cohorts: cohort 1 received oral atovaquone at the standard clinical dose of 750 mg twice daily, while cohort 2 did not. Primary imaging endpoint was change in tumor hypoxic volume (HV) measured by hypoxia PET-CT. Intercohort comparison of hypoxia gene expression signatures using RNA sequencing from resected tumors was performed., Results: Thirty patients were evaluable for hypoxia PET-CT analysis, 15 per cohort. Median treatment duration was 12 days. Eleven (73.3%) atovaquone-treated patients had meaningful HV reduction, with median change -28% [95% confidence interval (CI), -58.2 to -4.4]. In contrast, median change in untreated patients was +15.5% (95% CI, -6.5 to 35.5). Linear regression estimated the expected mean HV was 55% (95% CI, 24%-74%) lower in cohort 1 compared with cohort 2 ( P = 0.004), adjusting for cohort, tumor volume, and baseline HV. A key pharmacodynamics endpoint was reduction in hypoxia-regulated genes, which were significantly downregulated in atovaquone-treated tumors. Data from multiple additional measures of tumor hypoxia and perfusion are presented. No atovaquone-related adverse events were reported., Conclusions: This is the first clinical evidence that targeting tumor mitochondrial metabolism can reduce hypoxia and produce relevant antitumor effects at the mRNA level. Repurposing atovaquone for this purpose may improve treatment outcomes for NSCLC., (©2021 American Association for Cancer Research.)

T-LAK-originated protein kinase (TOPK) overexpression is a feature of multiple cancers, yet is absent from most phenotypically normal tissues. As such, TOPK expression profiling and the development of TOPK-targeting pharmaceutical agents have raised hopes for its future potential in the development of targeted therapeutics. Results presented in this paper confirm the value of TOPK as a potential target for the treatment of solid tumours, and demonstrate the efficacy of a TOPK inhibitor (OTS964) when used in combination with radiation treatment. Using H460 and Calu-6 lung cancer xenograft models, we show that pharmaceutical inhibition of TOPK potentiates the efficacy of fractionated irradiation. Furthermore, we provide in vitro evidence that TOPK plays a hitherto unknown role during S phase, showing that TOPK depletion increases fork stalling and collapse under conditions of replication stress and exogenous DNA damage. Transient knockdown of TOPK was shown to impair recovery from fork stalling and to increase the formation of replication-associated single-stranded DNA foci in H460 lung cancer cells. We also show that TOPK interacts directly with CHK1 and Cdc25c, two key players in the checkpoint signalling pathway activated after replication fork collapse. This study thus provides novel insights into the mechanism by which TOPK activity supports the survival of cancer cells, facilitating checkpoint signalling in response to replication stress and DNA damage.

Platinum chemotherapies are highly effective cytotoxic agents but often induce resistance when used as monotherapies. Combinatorial strategies limit this risk and provide effective treatment options for many cancers. Here, we repurpose atovaquone (ATQ), a well-tolerated & FDA-approved anti-malarial agent by demonstrating that it potentiates cancer cell death of a subset of platinums. We show that ATQ in combination with carboplatin or cisplatin induces striking and repeatable concentration- and time-dependent cell death sensitization in vitro across a variety of cancer cell lines. ATQ induces mitochondrial reactive oxygen species (mROS), depleting intracellular glutathione (GSH) pools in a concentration-dependent manner. The superoxide dismutase mimetic MnTBAP rescues ATQ-induced mROS production and pre-loading cells with the GSH prodrug N-acetyl cysteine (NAC) abrogates the sensitization. Together, these findings implicate ATQ-induced oxidative stress as key mediator of the sensitizing effect. At physiologically achievable concentrations, ATQ and carboplatin furthermore synergistically delay the growth of three-dimensional avascular spheroids. Clinically, ATQ is a safe and specific inhibitor of the electron transport chain (ETC) and is concurrently being repurposed as a candidate tumor hypoxia modifier. Together, these findings suggest that ATQ is deserving of further study as a candidate platinum sensitizing agent., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s) 2020.)

Ionising radiation causes cell death through the induction of DNA damage, particularly double-stranded DNA (dsDNA) breaks. Evidence suggests that adenoviruses inhibit proteins involved in the DNA damage response (DDR) to prevent recognition of double-stranded viral DNA genomes as cellular dsDNA breaks. We hypothesise that combining adenovirus treatment with radiotherapy has the potential for enhancing tumour-specific cytotoxicity through inhibition of the DDR and augmentation of virus production. We show that EnAd, an Ad3/Ad11p chimeric oncolytic adenovirus currently being trialled in colorectal and other cancers, targets the DDR pathway at a number of junctures. Infection is associated with a decrease in irradiation-induced 53BP1 and Rad51 foci formation, and in total DNA ligase IV levels. We also demonstrate a radiation-associated increase in EnAd production in vitro and in a pilot in vivo experiment. Given the current limitations of in vitro techniques in assessing for synergy between these treatments, we adapted the plaque assay to allow monitoring of viral plaque size and growth and utilised the xCELLigence cell adhesion assay to measure cytotoxicity. Our study provides further evidence on the interaction between adenovirus and radiation in vitro and in vivo and suggests these have at least an additive, and possibly a synergistic, impact on cytotoxicity.

Background: Enhancer of zeste 2 (EZH2) promotes prostate cancer progression. We hypothesized that increased EZH2 expression is associated with postradiotherapy metastatic disease recurrence, and may promote radioresistance., Methods: EZH2 expression was investigated using immunohistochemistry in diagnostic prostate biopsies of 113 prostate cancer patients treated with radiotherapy with curative intent. Associations between EZH2 expression in malignant and benign tissue in prostate biopsy cores and outcomes were investigated using univariate and multivariate Cox regression analyses. LNCaP and PC3 cell radiosensitivity was investigated using colony formation and γH2AX assays following UNC1999 chemical probe-mediated EZH2 inhibition., Results: While there was no significant association between EZH2 expression and biochemical recurrence following radiotherapy, univariate analysis revealed that prostate cancer cytoplasmic and total EZH2 expression were significantly associated with metastasis development postradiotherapy (P = 0.034 and P = 0.003, respectively). On multivariate analysis, the prostate cancer total EZH2 expression score remained statistically significant (P = 0.003), while cytoplasmic EZH2 expression did not reach statistical significance (P = 0.053). No association was observed between normal adjacent prostate EZH2 expression and biochemical recurrence or metastasis. LNCaP and PC3 cell treatment with UNC1999 reduced histone H3 lysine 27 tri-methylation levels. Irradiation of LNCaP or PC3 cells with a single 2 Gy fraction with UNC1999-mediated EZH2 inhibition resulted in a statistically significant, though modest, reduction in cell colony number for both cell lines. Increased γH2AX foci were observed 24 hours after ionizing irradiation in LNCaP cells, but not in PC3, following UNC1999-mediated EZH2 inhibition vs controls., Conclusions: Taken together, these results reveal that high pretreatment EZH2 expression in prostate cancer in diagnostic biopsies is associated with an increased risk of postradiotherapy metastatic disease recurrence, but EZH2 function may only at most play a modest role in promoting prostate cancer cell radioresistance., (© 2019 The Authors. The Prostate Published by Wiley Periodicals, Inc.)

Background: Pre-clinically, phosphoinositide 3-kinase (PI3K) inhibition radiosensitises tumours by increasing intrinsic radiosensitivity and by reducing tumour hypoxia. We assessed whether buparlisib, a class 1 PI3K inhibitor, can be safely combined with radiotherapy in patients with non-small cell lung carcinoma (NSCLC) and investigated its effect on tumour hypoxia., Methods: This was a 3 + 3 dose escalation and dose expansion phase I trial in patients with advanced NSCLC. Buparlisib dose levels were 50 mg, 80 mg and 100 mg once daily orally for 2 weeks, with palliative thoracic radiotherapy (20 Gy in 5 fractions) delivered during week 2. Tumour hypoxic volume (HV) was measured using 18 F-fluoromisonidazole positron-emission tomography-computed tomography at baseline and following 1 week of buparlisib., Results: Twenty-one patients were recruited with 9 patients evaluable for maximum tolerated dose (MTD) analysis. No dose-limiting toxicity was reported; therefore, 100 mg was declared the MTD, and 10 patients received this dose in the expansion phase. Ninety-four percent of treatment-related adverse events were ≤grade 2 with fatigue (67%), nausea (24%) and decreased appetite (19%) most common per patient. One serious adverse event (grade 3 hypoalbuminaemia) was possibly related to buparlisib. No unexpected radiotherapy toxicity was reported. Ten (67%) of 15 patients evaluable for imaging analysis were responders with 20% median reduction in HV at the MTD., Conclusion: This is the first clinical trial to combine a PI3K inhibitor with radiotherapy in NSCLC and investigate the effects of PI3K inhibition on tumour hypoxia. This combination was well tolerated and PI3K inhibition reduced hypoxia, warranting investigation into whether this novel class of radiosensitisers can improve radiotherapy outcomes., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

'Targeted' or 'biological' cancer treatments rely on differential gene expression between normal tissue and cancer, and genetic changes that render tumour cells especially sensitive to the agent being applied. Problems exist with the application of many agents as a result of damage to local tissues, tumour evolution and treatment resistance, or through systemic toxicity. Hence, there is a therapeutic need to uncover specific clinical targets which enhance the efficacy of cancer treatment whilst minimising the risk to healthy tissues. T-LAK cell-originated protein kinase (TOPK) is a MAPKK-like kinase which plays a role in cell cycle regulation and mitotic progression. As a consequence, TOPK expression is minimal in differentiated cells, although its overexpression is a pathophysiological feature of many tumours. Hence, TOPK has garnered interest as a cancer-specific biomarker and biochemical target with the potential to enhance cancer therapy whilst causing minimal harm to normal tissues. Small molecule inhibitors of TOPK have produced encouraging results as a stand-alone treatment in vitro and in vivo, and are expected to advance into clinical trials in the near future. In this review, we present the current literature pertaining to TOPK as a potential clinical target and describe the progress made in uncovering its role in tumour development. Firstly, we describe the functional role of TOPK as a pro-oncogenic kinase, followed by a discussion of its potential as a target for the treatment of cancers with high-TOPK expression. Next, we provide an overview of the current preclinical progress in TOPK inhibitor discovery and development, with respect to future adaptation for clinical use.

Cyclin-dependent kinase 1 (CDK1) orchestrates the transition from the G2 phase into mitosis and as cancer cells often display enhanced CDK1 activity, it has been proposed as a tumor specific anti-cancer target. Here we show that the effects of CDK1 inhibition are not restricted to tumor cells but can also reduce viability in non-cancer cells and sensitize them to radiation in a cell cycle dependent manner. Radiosensitization by the specific CDK1 inhibitor, RO-3306, was determined by colony formation assays in three tumor lines (HeLa, T24, SQ20B) and three non-cancer lines (HFL1, MRC-5, RPE). Initial results showed that CDK1 inhibition radiosensitized tumor cells, but did not sensitize normal fibroblasts and epithelial cells in colony formation assays despite effective inhibition of CDK1 signaling. Further investigation showed that normal cells were less sensitive to CDK1 inhibition because they remained predominantly in G1 for a prolonged period when plated in colony formation assays. In contrast, inhibiting CDK1 a day after plating, when the cells were going through G2/M phase, reduced their clonogenic survival both with and without radiation. Our finding that inhibition of CDK1 can damage normal cells in a cell cycle dependent manner indicates that targeting CDK1 in cancer patients may lead to toxicity in normal proliferating cells. Furthermore, our finding that cell cycle progression becomes easily stalled in non-cancer cells under normal culture conditions has general implications for testing anti-cancer agents in these cells.

Background: Tumour-specific radiosensitising treatments may enhance the efficacy of radiotherapy without exacerbating side effects. In this study we determined the radiation response following depletion or inhibition of TOPK, a mitogen-activated protein kinase kinase family Ser/Thr protein kinase that is upregulated in many cancers., Methods: Radiation response was studied in a wide range of cancer cell lines and normal cells using colony formation assays. The effect on cell cycle progression was assessed and the relationship between TOPK expression and therapeutic efficacy was studied in a cohort of 128 prostate cancer patients treated with radical radiotherapy., Results: TOPK knockdown did not alter radiation response in normal tissues, but significantly enhanced radiosensitivity in cancer cells. This result was recapitulated in TOPK knockout cells and with the TOPK inhibitor, OTS964. TOPK depletion altered the G 1 /S transition and G 2 /M arrest in response to radiation. Furthermore, TOPK depletion increased chromosomal aberrations, multinucleation and apoptotic cell death after irradiation. These results suggest a possible role for TOPK in the radiation-induced DNA damage checkpoints. These findings have clinical relevance, as elevated TOPK protein expression was associated with poorer clinical outcomes in prostate cancer patients treated with radical radiotherapy., Conclusions: This study demonstrates that TOPK disruption may cause tumour-specific radiosensitisation in multiple different tumour types.

Objective: To investigate mitophagy in 5 patients with severe dominantly inherited optic atrophy (DOA), caused by depletion of OPA1 (a protein that is essential for mitochondrial fusion), compared with healthy controls., Methods: Patients with severe DOA (DOA plus) had peripheral neuropathy, cognitive regression, and epilepsy in addition to loss of vision. We quantified mitophagy in dermal fibroblasts, using 2 high throughput imaging systems, by visualizing colocalization of mitochondrial fragments with engulfing autophagosomes., Results: Fibroblasts from 3 biallelic OPA1(-/-) patients with severe DOA had increased mitochondrial fragmentation and mitochondrial DNA (mtDNA)-depleted cells due to decreased levels of OPA1 protein. Similarly, in siRNA-treated control fibroblasts, profound OPA1 knockdown caused mitochondrial fragmentation, loss of mtDNA, impaired mitochondrial function, and mitochondrial mislocalization. Compared to controls, basal mitophagy (abundance of autophagosomes colocalizing with mitochondria) was increased in (1) biallelic patients, (2) monoallelic patients with DOA plus, and (3) OPA1 siRNA-treated control cultures. Mitophagic flux was also increased. Genetic knockdown of the mitophagy protein ATG7 confirmed this by eliminating differences between patient and control fibroblasts., Conclusions: We demonstrated increased mitophagy and excessive mitochondrial fragmentation in primary human cultures associated with DOA plus due to biallelic OPA1 mutations. We previously found that increased mitophagy (mitochondrial recycling) was associated with visual loss in another mitochondrial optic neuropathy, Leber hereditary optic neuropathy (LHON). Combined with our LHON findings, this implicates excessive mitochondrial fragmentation, dysregulated mitophagy, and impaired response to energetic stress in the pathogenesis of mitochondrial optic neuropathies, potentially linked with mitochondrial mislocalization and mtDNA depletion., (Copyright © 2016 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Colony formation is the gold standard assay for determining reproductive cell death after radiation treatment, since effects on proliferation often do not reflect survival. We have developed a high-throughput radiosensitivity screening method based on clonogenicity and screened a siRNA library against kinases. Thiamine pyrophosphokinase-1 (TPK1), a key component of Vitamin B1/thiamine metabolism, was identified as a target for radiosensitization. TPK1 knockdown caused significant radiosensitization in cancer but not normal tissue cell lines. Other means of blocking this pathway, knockdown of thiamine transporter-1 (THTR1) or treatment with the thiamine analogue pyrithiamine hydrobromide (PyrH) caused significant tumor specific radiosensitization. There was persistent DNA damage in cells irradiated after TPK1 and THTR1 knockdown or PyrH treatment. Thus this screen allowed the identification of thiamine metabolism as a novel radiosensitization target that affects DNA repair. Short-term modulation of thiamine metabolism could be a clinically exploitable strategy to achieve tumor specific radiosensitization.

The ataxia telangiectasia and Rad3-related (ATR) plays an important role in maintaining genome integrity during DNA replication through the phosphorylation and activation of Chk1 and regulation of the DNA damage response. Preclinical studies have shown that disruption of ATR pathway can exacerbate the levels of replication stress in oncogene-driven murine tumors to promote cell killing. Additionally, inhibition of ATR can sensitise tumor cells to radiation or chemotherapy. Accumulating evidence suggests that targeting ATR can selectively sensitize cancer cells but not normal cells to DNA damage. Furthermore, in hypoxic conditions, ATR blockade results in overloading replication stress and DNA damage response causing cell death. Despite the attractiveness of ATR inhibition in the treatment of cancer, specific ATR inhibitors have remained elusive. In the last two years however, selective ATR inhibitors suitable for in vitro and - most recently - in vivo studies have been identified. In this article, we will review the literature on ATR function, its role in DDR and the potential of ATR inhibition to enhance the efficacy of radiation and chemotherapy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

DNA polymerase eta (pol η) is the only DNA polymerase causally linked to carcinogenesis in humans. Inherited deficiency of pol η in the variant form of xeroderma pigmentosum (XPV) predisposes to UV-light-induced skin cancer. Pol η-deficient cells demonstrate increased sensitivity to cisplatin and oxaliplatin chemotherapy. We have found that XP30R0 fibroblasts derived from a patient with XPV are more resistant to cell kill by ionising radiation (IR) than the same cells complemented with wild-type pol η. This phenomenon has been confirmed in Burkitt's lymphoma cells, which either expressed wild-type pol η or harboured a pol η deletion. Pol η deficiency was associated with accumulation of cells in S-phase, which persisted after IR. Cells deficient in pol η demonstrated increased homologous recombination (HR)-directed repair of double strand breaks created by IR. Depletion of the HR protein, X-ray repair cross-complementing protein 3 (XRCC3), abrogated the radioresistance observed in pol η-deficient cells as compared with pol η-complemented cells. These findings suggest that HR mediates S-phase-dependent radioresistance associated with pol η deficiency. We propose that pol η protein levels in tumours may potentially be used to identify patients who require treatment with chemo-radiotherapy rather than radiotherapy alone for adequate tumour control.

DNA damaging agents such as radiotherapy and gemcitabine are frequently used for the treatment of pancreatic cancer. However, these treatments typically provide only modest benefit. Improving the low survival rate for pancreatic cancer patients therefore remains a major challenge in oncology. Inhibition of the key DNA damage response kinase ATR has been suggested as an attractive approach for sensitization of tumor cells to DNA damaging agents, but specific ATR inhibitors have remained elusive. Here we investigated the sensitization potential of the first highly selective and potent ATR inhibitor, VE-821, in vitro. VE-821 inhibited radiation- and gemcitabine-induced phosphorylation of Chk1, confirming inhibition of ATR signaling. Consistently, VE-821 significantly enhanced the sensitivity of PSN-1, MiaPaCa-2 and primary PancM pancreatic cancer cells to radiation and gemcitabine under both normoxic and hypoxic conditions. ATR inhibition by VE-821 led to inhibition of radiation-induced G 2/M arrest in cancer cells. Reduced cancer cell radiosurvival following treatment with VE-821 was also accompanied by increased DNA damage and inhibition of homologous recombination repair, as evidenced by persistence of γH2AX and 53BP1 foci and inhibition of Rad51 foci, respectively. These findings support ATR inhibition as a novel approach to improve the efficacy and therapeutic index of standard cancer treatments across a large proportion of pancreatic cancer patients.

Depletion of POLQ (DNA polymerase theta) has recently been shown to render tumour cells more sensitive to radiotherapy whilst having little or no effect on normal tissues. This finding led us to investigate whether tumours that overexpress POLQ are associated with an adverse outcome. We therefore correlated the clinical outcomes of two retrospective series of patients with early breast cancer with the expression levels of POLQ, as determined by microarray gene expression analysis. We found that a significant number of tumours overexpressed POLQ and that overexpression was correlated with ER negative disease (p=0.047) and high tumour grade (p=0.004), both of which are associated with poor clinical outcomes. POLQ overexpression was associated with poor relapse free survival rates on both univariate (HR 5.80; 95% CI, 2.220 to 15.159; p<0.001) and multivariate analysis (HR 8.086; 95% CI 2.340 to 27.948 p=0.001). Analysis of other published clinical series confirmed that POLQ overexpression is associated with adverse clinical outcomes. The poor prognosis associated with POLQ is independent of other clinical or pathological features. The mechanism that causes this adverse outcome remains to be elucidated but may in part arise from resistance to adjuvant treatment. These findings, combined with the limited normal tissue expression of POLQ, make it a very appealing target for possible clinical exploitation.

The effectiveness of radiotherapy treatment could be significantly improved if tumor cells could be rendered more sensitive to ionizing radiation (IR) without altering the sensitivity of normal tissues. However, many of the key therapeutically exploitable mechanisms that determine intrinsic tumor radiosensitivity are largely unknown. We have conducted a small interfering RNA (siRNA) screen of 200 genes involved in DNA damage repair aimed at identifying genes whose knockdown increased tumor radiosensitivity. Parallel siRNA screens were conducted in irradiated and unirradiated tumor cells (SQ20B) and irradiated normal tissue cells (MRC5). Using gammaH2AX foci at 24 hours after IR, we identified several genes, such as BRCA2, Lig IV, and XRCC5, whose knockdown is known to cause increased cell radiosensitivity, thereby validating the primary screening end point. In addition, we identified POLQ (DNA polymerase ) as a potential tumor-specific target. Subsequent investigations showed that POLQ knockdown resulted in radiosensitization of a panel of tumor cell lines from different primary sites while having little or no effect on normal tissue cell lines. These findings raise the possibility that POLQ inhibition might be used clinically to cause tumor-specific radiosensitization.

Cell signaling initiated at the epidermal growth factor receptor (EGFR), RAS oncoproteins, or PI3K contributes to a common pathway that promotes tumor survival after radiation-induced DNA damage. Inhibition of signaling at the level of EGFR, RAS, and PI3K has been tested, but clinical applicability has been shown only at the level of the EGFR or by inhibiting RAS indirectly with prenyltransferase inhibitors. Inhibition of PI3K with LY294002 or wortmannin lacks specificity and has shown unacceptable toxicity in preclinical studies. We previously showed that inhibiting class I PI3K expression with siRNA resulted in enhanced radiation killing of tumor cells. Here, we tested the possibility of achieving specific tumor cell radiosensitization with a pharmacologic inhibitor of class I PI3K, the pyridinylfuranopyrimidine inhibitor PI-103. Our results show that inhibiting PI3K activity reduces phosphorylation of AKT at serine 473. Reduced survival is seen in cells with AKT activation and seems preferential for tumor cells over cells in which AKT activity is not elevated. Reduced survival is accompanied by persistence of DNA damage as evidenced by persistence of gamma H2AX and Rad 51 foci after irradiation in the presence of the inhibitor. Reduced survival does not result from cell cycle redistribution during the PI-103 treatment intervals tested, although combining PI-103 treatment with radiation enhances the G(2)-M delay observed after irradiation. These results indicate that pharmacologic inhibitors with enhanced specificity for class I PI3K may be of benefit when combined with radiotherapy.

The hyaluronan receptor LYVE-1 is selectively expressed in the endothelium of lymphatic capillaries, where it has been proposed to function in hyaluronan clearance and hyaluronan-mediated leukocyte adhesion. However, recent studies suggest that hyaluronan homeostasis is unperturbed in LYVE-1(-/-) mice and that lymphatic adhesion/transmigration may be largely mediated by ICAM-1 and VCAM-1 rather than LYVE-1. Here we have explored the possibility that LYVE-1 functions during inflammation and report that the receptor is down-regulated by pro-inflammatory cytokines. Using cultured primary lymphatic endothelial cells, we show that surface expression of LYVE-1 is rapidly and reversibly lost after exposure to tumor necrosis factor-alpha (TNFalpha) and TNFbeta via internalization and degradation of the receptor in lysosomes, coupled with a shutdown in gene expression. Curiously, internalization does not result in significant uptake of hyaluronan, a process that is largely insensitive to the novel LYVE-1 adhesion blocking monoclonal antibody 3A, and proceeds almost equally in resting and inflammation-activated lymphatic endothelial cells. Finally, we show that TNF can induce down-modulation of LYVE-1 in ex vivo murine dermal tissue explants and present evidence that the process occurs in vivo, in the context of murine allergen-induced skin inflammation. These findings suggest that LYVE-1 can function independently of hyaluronan and have implications for the use of LYVE-1 as a histological marker for lymphangiogenesis in human pathology.

CCL21 is a homeostatic lymphoid chemokine instrumental in the recruitment and organization of T cells and dendritic cells into lymphoid T areas. In human secondary lymphoid organs (SLOs), CCL21 is produced by cells distributed throughout the T zone, whereas high endothelial venules (HEVs) lack CCL21 mRNA. A critical question remains whether the development of ectopic lymphoid tissue (ELT) in chronic inflammation recapitulates the features of SLOs. Thus, we systematically investigated in situ the cellular sources of CCL21 in SLOs and ELTs in several human diseases characterized by lymphoid neogenesis. By in situ hybridization and the use of combinatorial cell markers, we show that CCL21-producing vessels in inflamed tissues systematically display typical markers of lymphatic vessels, whereas, as in SLOs, ectopic HEVs do not synthesize detectable levels of CCL21. We also provide first-time evidence that a common pattern of CCL21 expression by CD45-negative myofibroblast-like cells localized in extra-HEV position and organized in a fibroblastic reticular network similarly characterizes human SLOs and organized ELTs. Altogether, our results demonstrate that in humans the pattern of CCL21 production in SLOs is maintained during inflammation and that the phenotypic and functional properties of stromal cells, found in SLO T-cell areas, are reproduced at ectopic sites.

The hyaluronan receptor LYVE-1 is expressed abundantly on the surfaces of lymphatic vessels and lymph node sinus endothelial cells from early development, where it has been suggested to function both in cell adhesion/transmigration and as a scavenger for hyaluronan turnover. To investigate the physiological role(s) of LYVE-1, we generated mice in which the gene for the receptor was inactivated by replacement with a beta-galactosidase reporter. LYVE-1(-/-) mice displayed an apparently normal phenotype, with no obvious alteration in lymphatic vessel ultrastructure or function and no apparent change in secondary lymphoid tissue structure or cellularity. In addition, the levels of hyaluronan in tissue and blood were unchanged. LYVE-1(-/-) mice also displayed normal trafficking of cutaneous CD11c(+) dendritic cells to draining lymph nodes via afferent lymphatics and normal resolution of oxazolone-induced skin inflammation. Finally, LYVE-1(-/-) mice supported normal growth of transplanted B16F10 melanomas and Lewis lung carcinomas. These results indicate that LYVE-1 is not obligatory for normal lymphatic development and function and suggest either the existence of compensatory receptors or a role more specific than that previously envisaged.

The sinusoidal endothelia of liver, spleen, and lymph node are major sites for uptake and recycling of waste macromolecules through promiscuous binding to a disparate family of scavenger receptors. Among the most complex is stabilin-1, a large multidomain protein containing tandem fasciclin domains, epidermal growth factor-like repeats, and a C-type lectin-like hyaluronan-binding Link module, which functions as an endocytic receptor for acetylated low density lipoprotein and advanced glycation end products. Intriguingly, stabilin-1 has also been reported to mediate both homing of leukocytes across lymph node high endothelial venules and adhesion of metastatic tumor cells to peritumoral lymphatic vessels. Currently, however, it is not clear how stabilin-1 mediates these distinct functions. To address the issue, we have investigated the tissue and subcellular localization of stabilin-1 in detail and assessed the functional status of its Link module. We show that stabilin-1 is almost entirely intracellular in lymph node high endothelial venules, lymphatic sinus endothelium, and cultured endothelial cells but that a finite population, detectable only by fluorescent antibody or fluorescein-labeled (Fl)-acetylated low density lipoprotein uptake, cycles rapidly between the plasma membrane and EEA-1+ve (early endosome antigen 1) early endosomes. In addition, we show using full-length stabilin-1 cDNA and a stabilin-1/CD44 chimera in HeLa cells that intracellular targeting is influenced by the transmembrane domain/cytoplasmic tail, which contains a putative dileucine (DXXLL) Golgi to endosomal sorting signal. Finally, we provide evidence that the stabilin-1 Link domain binds neither hyaluronan nor other glycosaminoglycans. These properties support a role for stabilin-1 as a rapidly recycling scavenger receptor and argue against a role in cell adhesion or lymphocyte homing.

In the paracortex of lymph nodes, cellular immune responses are generated against antigens captured in peripheral tissues by dendritic cells (DCs). DC-SIGN (dendritic cell-specific ICAM-3 grabbing nonintegrin), a C-type lectin exclusively expressed by DCs, functions as an antigen receptor as well as an adhesion receptor. A functional homologue of DC-SIGN, L-SIGN (liver/lymph node-SIGN, also called DC-SIGN-related), is expressed by liver sinus endothelial cells. In lymph nodes, both DC-SIGN and L-SIGN are expressed. In this study, we analyzed the distribution of these two SIGN molecules in detail in both normal and immunoreactive lymph nodes. DC-SIGN is expressed by mature DCs in paracortical areas and in addition by DCs with an immature phenotype in the outer zones of the paracortex. L-SIGN expression was also detected in the outer zones on sinus endothelial cells characterized by their expression of the lymphatic endothelial markers LYVE-1 and CLEVER-1. During both cellular and humoral immune responses changes in the amount of DC-SIGN+ immature and mature DCs and L-SIGN+ endothelial cells were observed, indicating that the influx or proliferation of these cells is dynamically regulated.

Early metastasis to lymph nodes is a frequent complication in human breast cancer. However, the extent to which this depends on lymphangiogenesis or on invasion of existing lymph vessels remains controversial. Although proliferating intratumoural lymphatics that promote nodal metastasis have been demonstrated in experimental breast tumours overexpressing VEGF-C, it has yet to be determined whether the same phenomena occur in spontaneous human breast cancers. To address this important issue, the present study investigated the lymphatics in primary human breast carcinoma (75 cases of invasive ductal and lobular breast cancer) by quantitative immunohistochemical staining for the lymphatic endothelial hyaluronan receptor LYVE-1, the blood vascular marker CD34, and the nuclear proliferation marker pKi67. None of the breast carcinomas was found to contain dividing lymph vessels, even in areas of active haemangiogenesis. Furthermore, the majority of non-dividing lymph vessels were confined to the tumour periphery where their incidence was low and unrelated to tumour size, grade or nodal status; rather, their density was inversely correlated with tumour aggressiveness as assessed by macrophage density (p = 0.009), and blood microvessel density (p = 0.05, Spearman Rank), as well as with distance from the tumour edge. Finally, a proportion of the peritumoural lymphatics contained tumour emboli associated with hyaluronan, indicating a possible role for LYVE-1/hyaluronan interactions in lymphatic invasion or metastasis. These results suggest that naturally occurring breast carcinomas invade and destroy lymph vessels rather than promoting their proliferation; that breast tumour lymphangiogenesis may not always occur at physiological VEGF-C levels; and that nodal metastasis can proceed via pre-existing lymphatics., (Copyright 2003 John Wiley & Sons, Ltd.)

How tumors access and spread via the lymphatics is not understood. Although it is clear that dissemination via the blood system involves hemangiogenesis, it is uncertain whether tumors also induce lymphangiogenesis or simply invade existing peritumoral vessels. To address the issue we quantitated tumor lymph vessels in archival specimens of head and neck cancer by immunostaining for the recently described lymphatic endothelial marker LYVE-1, the vascular endothelial marker CD34, and the pKi67 proliferation marker, correlating lymph vessel density and proliferation index with clinical and pathological variables. Discrete "hotspots" of intratumoral small proliferating lymphatics were observed in all carcinomas, and a high intratumoral lymph vessel density was associated with neck node metastases (n = 23; P = 0.027) and an infiltrating margin of tumor invasion (P = 0.046) in the oropharyngeal subgroup. Quantitation of the lymphangiogenic growth factor vascular endothelial growth factor C by real-time PCR and immunohistochemistry revealed higher levels of mRNA in tumor tissue than in normal samples (n = 8; P = 0.017), but no obvious correlation with intratumoral lymphatics. Our results provide new evidence that proliferating lymphatics can occur in human cancers and may in some cases contribute to lymph node metastasis.

The glycosaminoglycan hyaluronan is a key substrate for cell migration in tissues during inflammation, wound healing, and neoplasia. Unlike other matrix components, hyaluronan (HA) is turned over rapidly, yet most degradation occurs not locally but within distant lymph nodes, through mechanisms that are not yet understood. While it is not clear which receptors are involved in binding and uptake of hyaluronan within the lymphatics, one likely candidate is the lymphatic endothelial hyaluronan receptor LYVE-1 recently described in our laboratory (Banerji, S., Ni, J., Wang, S., Clasper, S., Su, J., Tammi, R., Jones, M., and Jackson, D.G. (1999) J. Cell Biol. 144, 789-801). Here we present evidence that LYVE-1 is involved in the uptake of hyaluronan by lymphatic endothelial cells using a new murine LYVE-1 orthologue identified from the EST data base. We show that mouse LYVE-1 both binds and internalizes hyaluronan in transfected 293T fibroblasts in vitro and demonstrate using immunoelectron microscopy that it is distributed equally among the luminal and abluminal surfaces of lymphatic vessels in vivo. In addition, we show by means of specific antisera that expression of mouse LYVE-1 remains restricted to the lymphatics in homozygous knockout mice lacking a functional gene for CD44, the closest homologue of LYVE-1 and the only other Link superfamily HA receptor known to date. Together these results suggest a role for LYVE-1 in the transport of HA from tissue to lymph and imply that further novel hyaluronan receptors must exist that can compensate for the loss of CD44 function.

The growth of blood and lymphatic vasculature is mediated in part by secreted polypeptides of the vascular endothelial growth factor (VEGF) family. The prototype VEGF binds VEGF receptor (VEGFR)-1 and VEGFR-2 and is angiogenic, whereas VEGF-C, which binds to VEGFR-2 and VEGFR-3, is either angiogenic or lymphangiogenic in different assays. We used an adenoviral gene transfer approach to compare the effects of these growth factors in adult mice. Recombinant adenoviruses encoding human VEGF-C or VEGF were injected subcutaneously into C57Bl6 mice or into the ears of nude mice. Immunohistochemical analysis showed that VEGF-C upregulated VEGFR-2 and VEGFR-3 expression and VEGF upregulated VEGFR-2 expression at 4 days after injection. After 2 weeks, histochemical and immunohistochemical analysis, including staining for the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), the vascular endothelial marker platelet-endothelial cell adhesion molecule-1 (PECAM-1), and the proliferating cell nuclear antigen (PCNA) revealed that VEGF-C induced mainly lymphangiogenesis in contrast to VEGF, which induced only angiogenesis. These results have significant implications in the planning of gene therapy using these growth factors.

Metastasis is a frequent and lethal complication of cancer. Vascular endothelial growth factor-C (VEGF-C) is a recently described lymphangiogenic factor. Increased expression of VEGF-C in primary tumours correlates with dissemination of tumour cells to regional lymph nodes. However, a direct role for VEGF-C in tumour lymphangiogenesis and subsequent metastasis has yet to be demonstrated. Here we report the establishment of transgenic mice in which VEGF-C expression, driven by the rat insulin promoter (Rip), is targeted to beta-cells of the endocrine pancreas. In contrast to wild-type mice, which lack peri-insular lymphatics, RipVEGF-C transgenics develop an extensive network of lymphatics around the islets of Langerhans. These mice were crossed with Rip1Tag2 mice, which develop pancreatic beta-cell tumours that are neither lymphangiogenic nor metastatic. Double-transgenic mice formed tumours surrounded by well developed lymphatics, which frequently contained tumour cell masses of beta-cell origin. These mice frequently developed pancreatic lymph node metastases. Our findings demonstrate that VEGF-C-induced lymphangiogenesis mediates tumour cell dissemination and the formation of lymph node metastases.

Metastasis of breast cancer occurs primarily through the lymphatic system, and the extent of lymph node involvement is a key prognostic factor for the disease. Whereas the significance of angiogenesis for tumor progression has been well documented, the ability of tumor cells to induce the growth of lymphatic vessels (lymphangiogenesis) and the presence of intratumoral lymphatic vessels have been controversial. Using a novel marker for lymphatic endothelium, LYVE-1, we demonstrate here the occurrence of intratumoral lymphangiogenesis within human breast cancers after orthotopic transplantation onto nude mice. Vascular endothelial growth factor (VEGF)-C overexpression in breast cancer cells potently increased intratumoral lymphangiogenesis, resulting in significantly enhanced metastasis to regional lymph nodes and to lungs. The degree of tumor lymphangiogenesis was highly correlated with the extent of lymph node and lung metastases. These results establish the occurrence and biological significance of intratumoral lymphangiogenesis in breast cancer and identify VEGF-C as a molecular link between tumor lymphangiogenesis and metastasis.

Metastasis to local lymph nodes via the lymphatic vessels is a common step in the spread of solid tumors. To investigate the molecular mechanisms underlying the spread of cancer by the lymphatics, we examined the ability of vascular endothelial growth factor (VEGF)-D, a ligand for the lymphatic growth factor receptor VEGFR-3/Flt-4, to induce formation of lymphatics in a mouse tumor model. Staining with markers specific for lymphatic endothelium demonstrated that VEGF-D induced the formation of lymphatics within tumors. Moreover, expression of VEGF-D in tumor cells led to spread of the tumor to lymph nodes, whereas expression of VEGF, an angiogenic growth factor which activates VEGFR-2 but not VEGFR-3, did not. VEGF-D also promoted tumor angiogenesis and growth. Lymphatic spread induced by VEGF-D could be blocked with an antibody specific for VEGF-D. This study demonstrates that lymphatics can be established in solid tumors and implicates VEGF family members in determining the route of metastatic spread.

A 15-year old girl presented with a slowly progressive spastic paraparesis since the age of 12. Creatine kinase was slightly increased. Muscle biopsy carried out during tendon surgery for severe toe-walking showed 'myopathic' changes. Subsequent neurological evaluation and radiological studies revealed a large extradural arachnoid cyst extending from the 11th thoracic vertebra to the first lumbar vertebra. Her condition improved after operation. The 'myopathic' features turned out to be the result of chronic spinal compression. MRI is the method of choice to examine patients with non-hereditary progressive spastic paraparesis. Muscle biopsy and tendon surgery should not be performed, without careful neurological examination.

Early onset cerebellar ataxia with retained tendon reflexes (EOCA) is a clinical syndrome characterised by progressive cerebellar ataxia with an onset before the age of 25 years and a wide spectrum of associated features. It is distinguished from Friedreich's ataxia (FA) mainly by the preservation of tendon reflexes, a better prognosis, and the absence of GAA expansion in the frataxin gene. Although EOCA is thought to be a hereditary disorder with an autosomal recessive mode of inheritance, genetic heterogeneity might underlie the spectrum of clinical features. In this case report we describe a patient with EOCA accompanied by pes cavus, hammer toes and peripheral neuropathy. The patient's father did not have any ataxia, but had the same foot deformities as his daughter and a slight peripheral neuropathy. The possible relationship between these clinical features is discussed.

Objective: To investigate the diagnostic value of magnetic resonance imaging (MRI) in the detection of early sacroiliitis., Methods: Twenty-five consecutive HLA-B27 positive patients with inflammatory low back pain and < or = grade 2 unilateral sacroiliitis on conventional radiography (modified New York criteria) were studied. Erythrocyte sedimentation rate, C-reactive protein, plain radiography (PR), and MRI of the sacroiliac (SI) joints were obtained at study entry and PR of the SI joints after 3 years. Each radiograph and MR image set was interpreted independently. SI joints were scored according to the modified New York Criteria for radiological sacroiliitis. MRI scans were also scored for the presence of subchondral marrow edema. The relationship between > or = grade 2 sacroiliitis (by modified New York criteria for radiological sacroiliitis) shown on MRI and the subsequent development of > or = grade 2 sacroiliitis on PR after 3 years was investigated., Results: At study entry > or = grade 2 sacroiliitis was found on MRI in 36 of 50 SI joints. Edema was found in 20 of 50 SI joints. After 3 years > or = grade 2 sacroiliitis was found on PR in 21 of 44 SI joints. The positive predictive value of > or = grade 2 sacroiliitis on MRI for the development of > or = grade 2 sacroiliitis on PR after 3 years was 60%; sensitivity was 85% and specificity 47%., Conclusion: Our data suggest that MRI of the SI joints can be used to identify sacroiliitis earlier than PR.

A 23-year-old woman was admitted with headache, nausea, vomiting and blurred vision on the left side. Neurological examination showed ptosis with a complete internal and external ophthalmoplegia and a red fullness around the left orbita. Computed tomographic scanning of the brain revealed no abnormalities. As she improved on high doses of steroids a diagnosis of Tolosa-Hunt syndrome (THS) seemed to be indicated. However, magnetic resonance imaging (MRI) showed a lesion with intermediate signal intensity in the left cavernous sinus. Craniotomy was performed when symptoms of THS recurred. Histopathological examination revealed a meningioma with a papillary aspect and some mitoses. This case illustrates that: (1) THS is still a diagnosis by exclusion; (2) MRI and histopathological examination are important if there is any doubt about the diagnosis; and (3) also when there is no doubt, improvement after steroid therapy may be a diagnostic pitfall. Therefore, not only MRI but also orbital phlebography and angiography should seriously be considered.

Eleven patients with clinically definite MS and three healthy controls were investigated by magnetic resonance spectroscopic imaging. The data sets were analysed for all voxels containing white matter only. We classify these voxels in healthy controls as normal white matter (NWM), and in MS patients as normal-appearing white matter unaffected by MS lesions (NAWM) or white matter affected by MS lesions. The spectra belonging to the voxels were analysed for content of cholines, creatines and N-acetylaspartate (NAA), and compared as a group. It was found that lesions differ from white matter in chemical composition and, moreover, that normal-appearing white matter differs from healthy white matter. Specifically, levels of NAA are lower in patients. There seems to be a linear relation between the composition of white matter and the expanded disability status scale value for the patient. The presence of lactate could not be established, and no unambiguous differences were found between patients with relapsing-remitting and relapsing-progressive disease.

Objective: To study the prevalence and nature of sterile spondylodiscitis in patients suffering from ankylosing spondylitis., Design: Descriptive., Setting: Department of Rheumatology, Medisch Spectrum Twente, Enschede, the Netherlands., Method: Of all ankylosing spondylitis patients suffering from sterile spondylodiscitis, the medical histories and the radiological and histological findings were analysed., Results: Among about 400 patients, 6 cases of sterile spondylodiscitis were found; 4 men and 2 women. The mean time lapse between diagnosis of ankylosing spondylitis and onset of spondylodiscitis symptoms was 7 years; in I patient discitis was the first symptom. Discitis changed the nature of the backache: it worsened during exercise and improved on resting. There was no history of trauma. Symptomatic and asyptomatic radiological abnormalities were seen at the same time in 2 patients. Symptoms disappeared 3-36 months after start of conservative treatment. Histological examination was performed in 2 cases and showed inflammatory changes., Conclusion: Spondylodiscitis in ankylosing spondylitis has a highly variable presentation and in general a good prognosis. Our findings support an inflammatory rather than a traumatic pathogenesis.

We describe a 68-year-old woman who had suffered pain, swelling, heat, and redness in the region of both clavicles for the last 2 years. Her erythrocyte sedimentation rate was markedly elevated; tests for rheumatoid factor were negative. At surgical exploration, ankylosis of the sternoclavicular joints, especially on the left side, was found. Biopsy revealed chronic nonspecific inflammation with new bone formation, consistent with the diagnosis of sternocostoclavicular hyperostosis or pustulotic arthroosteitis.

A 73-year-old female with severe sciatica suffered from an aneurysm of the left internal iliac artery. At first minimal caudographic abnormalities suggested intervertebral disc herniation and lumbar root compression but at exploratory surgery the diagnosis had to be rejected. Once the correct diagnosis was established by echography, CT-scanning and angiography surgical treatment of the aneurysm resulted in complete recovery. Because sciatic nerve lesions due to aneurysms in the pelvic region very seldom occur and may be the cause of diagnostic confusion, symptoms and treatment of these aneurysms are discussed.