Potentiation of antitumor immunity by antibody-directed enzyme prodrug therapy

Antibody-directed enzyme prodrug therapy (ADEPT) has displayed antitumor activity in animal models and clinical trials. We examined whether antitumor immunity is generated during ADEPT by employing an immunoenzyme composed of the monoclonal antibody (MAb) RH1 conjugated to ␤-glucuronidase to target rat AS-30D hepatocellular carcinoma tumors. A glucuronide prodrug of p-hydroxyaniline mustard was used to treat malignant ascites after immunoenzyme localization at the cancer cells. ADEPT cured more than 96% of Sprague-Dawley rats bearing advanced malignant ascites, and all cured rats were protected from a lethal challenge of AS-30D cells. Immunization with radiation-killed AS-30D cells or AS-30D cells coated with immunoenzyme did not provide tumor protection. Likewise, ex vivo treatment of tumor cells by ADEPT before injection into rats did not protect against a tumor challenge. AS-30D and N1-S1 hepatocellular carcinoma cells but not unrelated syngeneic tumor cells were lysed by peritoneal exudate cells isolated from ADEPT-cured rats. Depletion of CD8 ؉ but not CD4 ؉ T cells or natural killer (NK) cells reduced the cytolytic activity of peritoneal lymphocytes. ADEPT did not cure tumor-bearing rats depleted of CD4 ؉ and CD8 ؉ T cells even though it was curative when given 7 days after tumor transplantation in rats with an intact immune system, indicating that ADEPT can synergize with host immunity to increase therapeutic efficacy. These results have important implications for the clinical application of ADEPT

The Affinity of Elongated Membrane-Tethered Ligands Determines Potency of T Cell Receptor Triggering

T lymphocytes are important mediators of adoptive immunity but the mechanism of T cell receptor (TCR) triggering remains uncertain. The interspatial distance between engaged T cells and antigen-presenting cells (APCs) is believed to be important for topological rearrangement of membrane tyrosine phosphatases and initiation of TCR signaling. We investigated the relationship between ligand topology and affinity by generating a series of artificial APCs that express membrane-tethered anti-CD3 scFv with different affinities (OKT3, BC3, and 2C11) in addition to recombinant class I and II pMHC molecules. The dimensions of membrane-tethered anti-CD3 and pMHC molecules were progressively increased by insertion of different extracellular domains. In agreement with previous studies, elongation of pMHC molecules or low-affinity anti-CD3 scFv caused progressive loss of T cell activation. However, elongation of high-affinity ligands (BC3 and OKT3 scFv) did not abolish TCR phosphorylation and T cell activation. Mutation of key amino acids in OKT3 to reduce binding affinity to CD3 resulted in restoration of topological dependence on T cell activation. Our results show that high-affinity TCR ligands can effectively induce TCR triggering even at large interspatial distances between T cells and APCs

Global incidence, prevalence, years lived with disability (YLDs), disability-adjusted life-years (DALYs), and healthy life expectancy (HALE) for 371 diseases and injuries in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

Background: Detailed, comprehensive, and timely reporting on population health by underlying causes of disability and premature death is crucial to understanding and responding to complex patterns of disease and injury burden over time and across age groups, sexes, and locations. The availability of disease burden estimates can promote evidence-based interventions that enable public health researchers, policy makers, and other professionals to implement strategies that can mitigate diseases. It can also facilitate more rigorous monitoring of progress towards national and international health targets, such as the Sustainable Development Goals. For three decades, the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) has filled that need. A global network of collaborators contributed to the production of GBD 2021 by providing, reviewing, and analysing all available data. GBD estimates are updated routinely with additional data and refined analytical methods. GBD 2021 presents, for the first time, estimates of health loss due to the COVID-19 pandemic. Methods: The GBD 2021 disease and injury burden analysis estimated years lived with disability (YLDs), years of life lost (YLLs), disability-adjusted life-years (DALYs), and healthy life expectancy (HALE) for 371 diseases and injuries using 100 983 data sources. Data were extracted from vital registration systems, verbal autopsies, censuses, household surveys, disease-specific registries, health service contact data, and other sources. YLDs were calculated by multiplying cause-age-sex-location-year-specific prevalence of sequelae by their respective disability weights, for each disease and injury. YLLs were calculated by multiplying cause-age-sex-location-year-specific deaths by the standard life expectancy at the age that death occurred. DALYs were calculated by summing YLDs and YLLs. HALE estimates were produced using YLDs per capita and age-specific mortality rates by location, age, sex, year, and cause. 95% uncertainty intervals (UIs) were generated for all final estimates as the 2·5th and 97·5th percentiles values of 500 draws. Uncertainty was propagated at each step of the estimation process. Counts and age-standardised rates were calculated globally, for seven super-regions, 21 regions, 204 countries and territories (including 21 countries with subnational locations), and 811 subnational locations, from 1990 to 2021. Here we report data for 2010 to 2021 to highlight trends in disease burden over the past decade and through the first 2 years of the COVID-19 pandemic. Findings: Global DALYs increased from 2·63 billion (95% UI 2·44–2·85) in 2010 to 2·88 billion (2·64–3·15) in 2021 for all causes combined. Much of this increase in the number of DALYs was due to population growth and ageing, as indicated by a decrease in global age-standardised all-cause DALY rates of 14·2% (95% UI 10·7–17·3) between 2010 and 2019. Notably, however, this decrease in rates reversed during the first 2 years of the COVID-19 pandemic, with increases in global age-standardised all-cause DALY rates since 2019 of 4·1% (1·8–6·3) in 2020 and 7·2% (4·7–10·0) in 2021. In 2021, COVID-19 was the leading cause of DALYs globally (212·0 million [198·0–234·5] DALYs), followed by ischaemic heart disease (188·3 million [176·7–198·3]), neonatal disorders (186·3 million [162·3–214·9]), and stroke (160·4 million [148·0–171·7]). However, notable health gains were seen among other leading communicable, maternal, neonatal, and nutritional (CMNN) diseases. Globally between 2010 and 2021, the age-standardised DALY rates for HIV/AIDS decreased by 47·8% (43·3–51·7) and for diarrhoeal diseases decreased by 47·0% (39·9–52·9). Non-communicable diseases contributed 1·73 billion (95% UI 1·54–1·94) DALYs in 2021, with a decrease in age-standardised DALY rates since 2010 of 6·4% (95% UI 3·5–9·5). Between 2010 and 2021, among the 25 leading Level 3 causes, age-standardised DALY rates increased most substantially for anxiety disorders (16·7% [14·0–19·8]), depressive disorders (16·4% [11·9–21·3]), and diabetes (14·0% [10·0–17·4]). Age-standardised DALY rates due to injuries decreased globally by 24·0% (20·7–27·2) between 2010 and 2021, although improvements were not uniform across locations, ages, and sexes. Globally, HALE at birth improved slightly, from 61·3 years (58·6–63·6) in 2010 to 62·2 years (59·4–64·7) in 2021. However, despite this overall increase, HALE decreased by 2·2% (1·6–2·9) between 2019 and 2021. Interpretation: Putting the COVID-19 pandemic in the context of a mutually exclusive and collectively exhaustive list of causes of health loss is crucial to understanding its impact and ensuring that health funding and policy address needs at both local and global levels through cost-effective and evidence-based interventions. A global epidemiological transition remains underway. Our findings suggest that prioritising non-communicable disease prevention and treatment policies, as well as strengthening health systems, continues to be crucially important. The progress on reducing the burden of CMNN diseases must not stall; although global trends are improving, the burden of CMNN diseases remains unacceptably high. Evidence-based interventions will help save the lives of young children and mothers and improve the overall health and economic conditions of societies across the world. Governments and multilateral organisations should prioritise pandemic preparedness planning alongside efforts to reduce the burden of diseases and injuries that will strain resources in the coming decades. Funding: Bill & Melinda Gates Foundation

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake

Global burden of 288 causes of death and life expectancy decomposition in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND Regular, detailed reporting on population health by underlying cause of death is fundamental for public health decision making. Cause-specific estimates of mortality and the subsequent effects on life expectancy worldwide are valuable metrics to gauge progress in reducing mortality rates. These estimates are particularly important following large-scale mortality spikes, such as the COVID-19 pandemic. When systematically analysed, mortality rates and life expectancy allow comparisons of the consequences of causes of death globally and over time, providing a nuanced understanding of the effect of these causes on global populations. METHODS The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 cause-of-death analysis estimated mortality and years of life lost (YLLs) from 288 causes of death by age-sex-location-year in 204 countries and territories and 811 subnational locations for each year from 1990 until 2021. The analysis used 56 604 data sources, including data from vital registration and verbal autopsy as well as surveys, censuses, surveillance systems, and cancer registries, among others. As with previous GBD rounds, cause-specific death rates for most causes were estimated using the Cause of Death Ensemble model-a modelling tool developed for GBD to assess the out-of-sample predictive validity of different statistical models and covariate permutations and combine those results to produce cause-specific mortality estimates-with alternative strategies adapted to model causes with insufficient data, substantial changes in reporting over the study period, or unusual epidemiology. YLLs were computed as the product of the number of deaths for each cause-age-sex-location-year and the standard life expectancy at each age. As part of the modelling process, uncertainty intervals (UIs) were generated using the 2·5th and 97·5th percentiles from a 1000-draw distribution for each metric. We decomposed life expectancy by cause of death, location, and year to show cause-specific effects on life expectancy from 1990 to 2021. We also used the coefficient of variation and the fraction of population affected by 90% of deaths to highlight concentrations of mortality. Findings are reported in counts and age-standardised rates. Methodological improvements for cause-of-death estimates in GBD 2021 include the expansion of under-5-years age group to include four new age groups, enhanced methods to account for stochastic variation of sparse data, and the inclusion of COVID-19 and other pandemic-related mortality-which includes excess mortality associated with the pandemic, excluding COVID-19, lower respiratory infections, measles, malaria, and pertussis. For this analysis, 199 new country-years of vital registration cause-of-death data, 5 country-years of surveillance data, 21 country-years of verbal autopsy data, and 94 country-years of other data types were added to those used in previous GBD rounds. FINDINGS The leading causes of age-standardised deaths globally were the same in 2019 as they were in 1990; in descending order, these were, ischaemic heart disease, stroke, chronic obstructive pulmonary disease, and lower respiratory infections. In 2021, however, COVID-19 replaced stroke as the second-leading age-standardised cause of death, with 94·0 deaths (95% UI 89·2-100·0) per 100 000 population. The COVID-19 pandemic shifted the rankings of the leading five causes, lowering stroke to the third-leading and chronic obstructive pulmonary disease to the fourth-leading position. In 2021, the highest age-standardised death rates from COVID-19 occurred in sub-Saharan Africa (271·0 deaths [250·1-290·7] per 100 000 population) and Latin America and the Caribbean (195·4 deaths [182·1-211·4] per 100 000 population). The lowest age-standardised death rates from COVID-19 were in the high-income super-region (48·1 deaths [47·4-48·8] per 100 000 population) and southeast Asia, east Asia, and Oceania (23·2 deaths [16·3-37·2] per 100 000 population). Globally, life expectancy steadily improved between 1990 and 2019 for 18 of the 22 investigated causes. Decomposition of global and regional life expectancy showed the positive effect that reductions in deaths from enteric infections, lower respiratory infections, stroke, and neonatal deaths, among others have contributed to improved survival over the study period. However, a net reduction of 1·6 years occurred in global life expectancy between 2019 and 2021, primarily due to increased death rates from COVID-19 and other pandemic-related mortality. Life expectancy was highly variable between super-regions over the study period, with southeast Asia, east Asia, and Oceania gaining 8·3 years (6·7-9·9) overall, while having the smallest reduction in life expectancy due to COVID-19 (0·4 years). The largest reduction in life expectancy due to COVID-19 occurred in Latin America and the Caribbean (3·6 years). Additionally, 53 of the 288 causes of death were highly concentrated in locations with less than 50% of the global population as of 2021, and these causes of death became progressively more concentrated since 1990, when only 44 causes showed this pattern. The concentration phenomenon is discussed heuristically with respect to enteric and lower respiratory infections, malaria, HIV/AIDS, neonatal disorders, tuberculosis, and measles. INTERPRETATION Long-standing gains in life expectancy and reductions in many of the leading causes of death have been disrupted by the COVID-19 pandemic, the adverse effects of which were spread unevenly among populations. Despite the pandemic, there has been continued progress in combatting several notable causes of death, leading to improved global life expectancy over the study period. Each of the seven GBD super-regions showed an overall improvement from 1990 and 2021, obscuring the negative effect in the years of the pandemic. Additionally, our findings regarding regional variation in causes of death driving increases in life expectancy hold clear policy utility. Analyses of shifting mortality trends reveal that several causes, once widespread globally, are now increasingly concentrated geographically. These changes in mortality concentration, alongside further investigation of changing risks, interventions, and relevant policy, present an important opportunity to deepen our understanding of mortality-reduction strategies. Examining patterns in mortality concentration might reveal areas where successful public health interventions have been implemented. Translating these successes to locations where certain causes of death remain entrenched can inform policies that work to improve life expectancy for people everywhere. FUNDING Bill & Melinda Gates Foundation

Elevated Topoisomerase I Activity in Cervical Cancer as a Target for Chemoradiation Therapy

Objective. The aim of this study was to determine whether the activity of topoisomerase I (topo I), the target of the anti-neoplastic drug camptothecin (CPT), is elevated in cervical cancer and whether CPT can radiosensitize cervical tumors. Methods. The topo I activity of 11 normal cervix and 30 cervical carcinoma tumors was assayed by measuring the relaxation of supercoiled DNA. Subconfluent or postconfluent CaSki human cervical carcinoma cells were exposed to CPT (1-5000 ng/ml) and immediately X-irradiated (0 -800 cGy). Cell survival was determined by clonogenic assay. Results. Mean topo I activity in cervical cancer (3.0 ؎ 0.06 h ؊1 ) was significantly greater than in normal cervix tissue (0.29 ؎ 0.06 h ؊1 ). Stage 3 and 4 cervical carcinoma specimens displayed a trend of greater topo I activity (5.88 ؎ 3.7 h ؊1 ) than stage 1 and 2 tumors (2.57 ؎ 0.47 h ؊1 ). No correlation between topo I protein levels and catalytic activity was found. Combined treatment of subconfluent CaSki cells with CPT and ionizing radiation resulted in additive killing of cells. Combined treatment of postconfluent CaSki cells with low doses of radiation (200 and 400 cGy) and 1 or 10 ng/ml CPT for 2 or 48 h produced significant cytotoxicity compared to CPT or radiation alone, which were ineffective at these doses. Conclusions. Topo I activity is elevated in cervical cancer compared to normal cervix. The radiosensitivity of noncycling cells within cervical tumors may be increased by simultaneous treatment with low doses of CPT or other topo I inhibitors. © 2000 Academic Press Key Words: camptothecin; topoisomerase I; cervical carcinoma; radiation; chemoradiation. INTRODUCTION Cervical carcinoma is one of the most common lethal malignancies affecting women, despite the widespread use of cervical cytological screening programs. It is estimated that 12,800 new cases of invasive carcinoma of the cervix and 4600 deaths related to this disease will occur in the United States in 2000 Radiotherapy is the standard, definitive therapy for advanced disease with cure rates ranging from 18 to 75% depending on the extent of disease Camptothecin (CPT) 2 and related analogs such as topotecan and CPT-11 are currently under investigation for cancer chemotherapy. Topotecan, a water-soluble derivative of CPT, has demonstrated activity against ovarian cancer in clinical trials Topo I is a nuclear enzyme that plays an important role in DNA replication and RNA transcription We have previously shown that camptothecin can potentiate the effects of radioimmunoconjugate therapy in a rat hepatoma ascites model MATERIALS AND METHODS Reagents and cells. Camptothecin and common biological reagents were purchased from Sigma Chemical Company (St. Louis, MO). Immunochemicals were purchased from Organon Teknika (Turnhout, Belgium). Camptothecin stock solutions were made in dimethylsulfoxide at 2.5 mg/ml, sterilized by filtration, and stored at Ϫ70°C. Spodoptera frugiperda cells were purchased from Pharmingen (San Diego, CA) and cultured according to the supplier's instructions. The CaSki human cervical carcinoma cell line was kindly provided by Dr. R. A. Pattillo, Medical College of Wisconsin, Milwaukee. HS 1025 and HS 1023 cervical carcinoma and H2669 melanoma cell lines were provided by Dr. Hellstrom, University of Washington, Seattle. TSGH 8302 cervical carcinoma cells were obtained from the Cancer Research Laboratory, Department of Medical Research, Tri-Service General Hospital, Taipei, Taiwan. HA22T human hepatocellular carcinoma cells were provided by Dr. C. P. Hu, Veteran's General Hospital, Taipei, Taiwan. All other cell lines were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI 1640 medium supplemented with 2.98 g/L Hepes, 2 g/L NaHCO 3 , 100 U/ml penicillin, 100 g/ml streptomycin, and 5% heat-inactivated bovine serum (culture medium). Tissues. Samples of cervical carcinoma and normal cervix were obtained from patients who underwent primary surgery at Tri-Service General Hospital, Taipei, Taiwan. Care was taken to ensure that normal and tumor components of samples were separated during tumor dissection. All specimens were immediately frozen in liquid nitrogen and stored at Ϫ135°C. The pathology of all tissues was microscopically confirmed by a pathologist. Nuclear extracts. Cells or finely diced tissues samples were washed once each with ice-cold PBS and nuclear buffer (150 mM NaCl, 1 mM KH 2 PO 4 , 5 mM MgCl 2 , 1 mM EGTA, 0.2 mM dithiothreitol, 10% (v/v) glycerol, and 0.1 mM PMSF, pH 6.4). Tissues or cells were suspended in nuclear buffer containing 0.3% Triton X-100 and disrupted in a mechanical dounce homogenizer on ice. Nuclei, collected by centrifugation at 1000g for 10 min, were washed once with nuclear buffer before the addition of nuclear buffer containing 0.25 M NaCl. Nuclei were gently rotated for 30 min at 4°C, centrifuged at 16,000g for 30 min to remove debris, and immediately assayed for topo I activity or stored at Ϫ135°C. At least two independent nuclear extracts were prepared for each sample. Topo I relaxation assay. Supercoiled pBR322 plasmid was purified from bacterial cultures by alkaline lysis followed by equilibration centrifugation in a cesium chloride-ethidium bromide continuous gradient for 36 h. Aliquots of supercoiled DNA were stored in absolute ethanol at Ϫ80°C. To assay topo I activity, 1.0 g supercoiled pBR322 was preheated to 37°C in 15 L of reaction buffer (10 mM Tris-HCl, pH 7.5, 200 mM KCl, 10 mM MgCl 2 , 1 mM EDTA, 1 mg/ml BSA, and 1 mM dithiothreitol) before the addition of 5 L nuclear extract diluted in reaction buffer. The reaction was terminated after 30 min by addition of 2 L of 10% SDS. Two microliters of loading dye (40% sucrose, 0.025% bromphenol blue, and 50 mM EDTA) was added and samples were electrophoresed at 3 V/cm for 5 h in a 1% TPE agarose gel containing 0.03% SDS CAMPTOTHECIN POTENTIATION OF RADIOTHERAPY to separate relaxed and supercoiled DNA. Gels were extensively washed with water to remove SDS and stained with ethidium bromide. Gel images were captured on an Eagle eye under UV illumination. The supercoiled DNA band was quantified on a Macintosh computer using the public domain NIH image program (developed at NIH and available at http:// rsb.info.nih.gov/nih-image/). Topo I activity was calculated as the number of micrograms pBR322 relaxed per microgram of nuclear extract per hour and has units of h Ϫ1 . Serial dilutions of each extract were assayed and only bands containing between 10 and 90% relaxed pBR322 DNA were used to calculate topo I activity. Each extract was assayed two to three times and the mean topo I activity was calculated. Production of recombinant human topo I. The 3645-base EcoRI cDNA fragment of human topo I present in a pUC9 plasmid (T1B, generously provided by Dr. Alastair Mackay, Department of Cell Biology and Anatomy, JHU Medical School, Baltimore, MD) was subcloned into the unique EcoRI site of the baculovirus transfer vector pVL1393 (Pharmingen) under the control of the polyhedrin promoter. A single recombinant baculovirus, produced in Sf21 cells according to the supplier's instructions (Pharmingen), was selected for production of human topo I. Sf21 cells were infected with virus at a MOI of 10 and cultured for 4 days at 27°C. Nuclear extracts were prepared from the cells and topo I was purified on Hitrap heparin and phenyl Sepharose CL-4B columns (Pharmacia) as described Generation of anti-topo I antibodies. A female rabbit was sc injected with 150 g of recombinant human topo I in complete Freund's adjuvant and boosted at 1-month intervals with 100 g of topo I in incomplete adjuvant. Blood was collected from the marginal ear vein 2 weeks after a final boost. Serum was collected and stored at Ϫ80°C. Immunoblots. Nuclear extracts were diluted in nucleus buffer to known concentrations and electrophoresed on a 3-12.5% gradient SDS-PAGE under reducing conditions. Proteins were transferred to nitrocellulose paper by passive diffusion as described Clonogenic assays. CPT cytotoxicity against subconfluent cells was determined in six-well plates by adding CPT for various periods to 150 -15,000 cells in culture medium. The cells were then washed twice with PBS to remove free drug and cultured for 6 days in clonogenic medium (culture medium supplemented with 5% conditioned medium, prepared from confluent cultures of the corresponding cell line). For postconfluent studies, cells were cultured in six-well plates until confluence was reached. The cells were then cultured for 1 week in medium containing 0.1% bovine serum. Three-fourths of the culture medium was refreshed daily. Under these conditions, greater than 95% of the cells were in the G 0 /G 1 phase of the cell cycle as determined by propidium iodide staining of DNA followed by flow cytometer analysis. After cells were exposed to CPT for 2 or 48 h in medium containing 0.1% bovine serum, the cells were washed twice with PBS and 100 to 10,000 cells were cultured in six-well culture plates in clonogenic medium for 6 days. For radiation studies, CaSki cells were irradiated in a Torrex 150D X-ray source (EG&G Astrophysics Research Corp., Long Beach, CA) at a rate of 160 cGy/min. Preconfluent CaSki cells were cultured in clonogenic medium for 6 days whereas postconfluent cells were plated in clonogenic medium at known cell concentrations 2 or 48 h after irradiation and cultured for 8 days. For combination studies, CPT was added immediately before X-ray irradiation. After 2 or 48 h, the cells were washed twice with PBS and incubated in clonogenic medium for 8 days. In some experiments, cells were exposed to 5 mM 3-aminobenzamide for 1 h before the addition of CPT and irradiation of the cells. Colonies were fixed and stained with 0.5% methylene blue in 50% ethanol/50% water (vol/vol) and visually counted under a microscope. Pre-and postconfluent CaSki cells had a plating efficiency of 40 -50% under these conditions. Cell survival is expressed as the percentage of survival relative to control cells that were not exposed to CPT or radiation. Isobolograms. Analysis of the effects of combination treatment in exponentially growing cells was performed by constructing isoeffect curves (isobolograms) as described by Steel and Peckham [26]. Mode I isoeffect curves were constructed by reading the doses of radiation or camptothecin from the respective single-agent dose-response curves, starting at zero dose of each agent, that added up to 20% cell survival. Mode II curves were formed in the same way except that the steepest portion of the radiation dose-response curve was used in the construction. Combination treatment results were plotted on the isobologram by interpolation of the combined doseresponse curves at 20% cell survival. Combinations resulting in points falling in the region to the left of the isoeffect curves correspond to positive or supra-additive interactions between the combined agents, points to the right of the curves represent subadditive effects, and combined doses falling in the region between the mode I and mode II curves are attributed to additive interactions of the single agents RESULTS Topoisomerase I Activity The topo I activity of nuclear extracts prepared from normal and tumor specimens was assayed by measuring the relaxation of supercoiled DNA. Densitometer quantitation of supercoiled DNA band intensity after electrophoresis of DNA on agarose gels allowed an estimation of topo I activity. Under the conditions employed in the assay, the intensity of pBR322 DNA standards was linearly related to DNA concentration from 0 to 1 g (data not shown). A range of concentrations was assayed for each nuclear extract. The addition of increasing amounts of nuclear extract produced increased relaxation of supercoiled pBR322 DNA to the fully relaxed form. Only band intensities corresponding to between 10 and 90% DNA relaxation were employed for calculation of topo I activity to prevent saturation of the assay and increase the assay precision. To examine topo I protein levels, polyclonal antibodies were generated against recombinant human topo I. Anti-topo I antiserum bound to topo I present in nuclear extracts prepared from CaSki cervical carcinoma cell

Effect of Cellular Location of Human Carboxylesterase 2 on CPT-11 Hydrolysis and Anticancer Activity.

CPT-11 is an anticancer prodrug that is clinically used for the treatment of metastatic colorectal cancer. Hydrolysis of CPT-11 by human carboxylesterase 2 (CE2) generates SN-38, a topoisomerase I inhibitor that is the active anti-tumor agent. Expression of CE2 in cancer cells is under investigation for the tumor-localized activation of CPT-11. CE2 is normally expressed in the endoplasmic reticulum of cells but can be engineered to direct expression of active enzyme on the plasma membrane or as a secreted form. Although previous studies have investigated different locations of CE2 expression in cancer cells, it remains unclear if CE2 cellular location affects CPT-11 anticancer activity. In the present study, we directly compared the influence of CE2 cellular location on substrate hydrolysis and CPT-11 cytotoxicity. We linked expression of CE2 and enhanced green fluorescence protein (eGFP) via a foot-and-mouth disease virus 2A (F2A) peptide to facilitate fluorescence-activated cell sorting to achieve similar expression levels of ER-located, secreted or membrane-anchored CE2. Soluble CE2 was detected in the medium of cells that expressed secreted and membrane-anchored CE2, but not in cells that expressed ER-retained CE2. Cancer cells that expressed all three forms of CE2 were more sensitive to CPT-11 as compared to unmodified cancer cells, but the membrane-anchored and ER-retained forms of CE2 were consistently more effective than secreted CE2. We conclude that expression of CE2 in the ER or on the membrane of cancer cells is suitable for enhancing CPT-11 anticancer activity