DAMPA is thought to be an inactive metabolite of MTX because it is not an effective inhibitor of the MTX target enzyme dihydrofolate reductase. DAMPA is eliminated more rapidly than MTX in these patients, which suggests a nonrenal route of elimination. In a phase II study (May 1997–March 2002), CPDG2 was administered intravenously to 82 patients at a median dose of 50 U kg−1 (range 33–60 U kg−1). Eligible patients for this study had serum MTX concentrations of >10 μM at 36 h or >5 μM at 42 h after start of MTX infusion and documented renal failure . Immediately before CPDG2 administration, a median MTX serum level of 11.93 μM (range 0.52–901 μM) was documented. Carboxypeptidase G2 was given at a median of 52 h (range 25–178 h) following the start of an MTX infusion of 1–12 g m−2 4–36 h−1 and resulted in a rapid 97% (range 73–99%) reduction of the MTX serum level. Toxicity related to CPDG2 was not observed. Toxicity related to MTX was documented in about half the patients; four patients died despite CPDG2 administration due to severe myelosuppression and septic complications. In conclusion, administration of CPDG2 is a well-tolerated, safe and a very effective way of MTX elimination in delayed excretion due to renal failure.The methotrexate (MTX) rescue agent carboxypeptidase G It may cause acute nephrotoxicity, which is presumed to result from its precipitation or its relatively insoluble metabolites in acidic urine. This nephrotoxicity leads to delayed MTX elimination, ineffective rescue and a marked increase of other nonhaematological and haematological toxicities associated with MTX, such as myelosuppression, oro-gastrointestinal mucositis and dermatitis. In early studies using HD-MTX, severe toxicity occurred in approximately 10% of patients and there was a 6% mortality rate . HoweverμM, high doses of LV are not likely to completely reverse the toxicity of MTX  was originally extracted from Pseudomonas stutzeri. During the 1970s, CPDG1 was administered successfully to a small number of patients with brain tumours and to one patient with pre-existing renal failure after MTX administration. However, the bacterial source for CPDG1 is no longer available methyl]-methylamino]-benzoic acid) and glutamate. In contrast to CPDG1, CPDG2 has a higher affinity for MTX (Km 8 μM) than LV (Km 120 μM) and 5-methyltetrahydrofolate (Km 35 μM), the primary circulating metabolite of LV. In studies performed in rhesus monkeys, a single CPDG2 dose of 50 units (U) kg−1 lowered serum MTX steady-state concentration from 10 μM to nontoxic levels of <0.05 μM within 30 min  resulted in a rapid 95–99% reduction of plasma levels of MTX in patients with renal dysfunction after HD-MTX .The study was a prospective, open, nonrandomised, multicentre trial. The primary study centres were in Bonn, Berlin and Vienna. Their task consisted of consultation and emergency dispatch of CPDGμM at 36 h or of >5 μM at 42 h or of >3 μM at 48 h after start of MTX infusion, and (2) decreased diuresis (less than 50% excretion of the input hydration) or (3) a serum creatinine (CreaS) >1.5 times the upper limit of normal and documented increase of CreaS during the infusion period.Patients of any age were eligible if they had (1) serum MTX concentration of >10 2 was manufactured by the Centre for Applied Microbiology and Research  and supplied in lyophilised form with vials containing 1000 U of enzyme activity. The vials were each resuspended in 1 ml of sterile isotonic saline; further dilution with sterile isotonic saline (1 : 5 or 1 : 10) was recommended. Carboxypeptidase G2 was administered at a dose of 50 U kg−1 over 5 min intravenously by an infusion pump or by bolus injection. Following each dose of CPDG2, serum MTX concentrations were determined. Patients who experienced a decrease in serum MTX concentration greater than one logarithm following CPDG2 administration but still had serum MTX concentrations >1 μM might receive additional doses of CPDG2 with the approval of the principal investigator of the study.Recombinant CPDG2 and readministered 1 h following enzyme injection. The recommended LV dose during the first 24 h after the CPDG2 injection was 100 mg m−2 every 6 h followed by an increased rescue for 5 days according to the scheme recommended in the ALL BFM 95 protocol  were determined according to National Cancer Institute Common Toxicity Criteria (CTC) version 2.0.Patients were evaluated daily for signs and symptoms of MTX toxicity. Complete blood counts with differential, bilirubin and ALT, AST were determined at least twice weekly. Recovery of renal function was documented daily by serum creatinine. The nonhaematological  and haematological toxicity of MTX and side effects of CPDG2, and then once daily until recovery of kidney function and decrease of MTX serum levels to less than 0.1 μM. Most of the local hospitals determined the MTX level immediately with the methods such as fluorescence polarisation immunoassay (TDX) or enzyme-multiplied immunotechnique (EMIT). In a few centres, additional blood samples for analysis by high-pressure liquid chromatography (HPLC) were placed on ice, and serum was rapidly separated by centrifugation. To inactivate CPDG2, the serum samples were heated to greater than 80°C for 5 min in a water bath or treated with 1 N HCl to give a final concentration of 0.1 N hydrochloric acid. If the enzyme activity in the samples was not fully stopped, MTX in the sample might continue to be degraded after collection. Serum was stored at −20°C until time of analysis. All serum samples were sent on dry ice to a central laboratory where they were stored at −20°C until analysis by HPLC.EDTA samples for MTX determination were obtained immediately before and 15, 30, 60 and 120 min following the dose of CPDG2 action on MTX, is known to crossreact with MTX in most commercial immunological MTX assays ), samples were injected onto a 3 μm, Supelcosil™ LC-18-DB 15 cm × 4.6 mm  radial compression analytical column with a Supelquard™ LC-18-DB 5 μm  guard column and eluted isocratically with 80 : 20 (v v−1) 0.1 mol l−1 sodium phosphate (pH 6.8) : methanol (Lichrosolv®) at a flow rate of 1.2 ml min−1. Eluent was monitored using a Waters 490 UV adjustable wavelength detector at a wavelength of 310 nm .Serum MTX was measured by HPLC using a modification of a previously reported method methyl]amino]-benzoic acid (Dm-APA) were approximately 5.6, 9.5 and 3.5 min, respectively. The peak areas were evaluated using the Chromquest software . Methotrexate and DAMPA were corrected by means of an internal standard in combination with a calibration curve.2 and (3) for the toxicity of MTX due to complete data documentation. Data were evaluated using descriptive statistical methods .Patient data were collected consecutively, recorded on data sheets prepared centrally beforehand, checked by the principal investigator and recorded in a protected database at the University of Bonn. The biometric analysis was performed for all eligible patients who were evaluable (1) for response to the study medication, (2) for the toxicity of CPDG2 and had given their written informed consent.The study was conducted in accordance with the updated declaration of Helsinki  and approved by the local ethics committees in Bonn, Berlin and Vienna. Prior to enrolment in the study, the patient's parents/legal representatives and/or the patients themselves were informed of the investigational character of the study and the emergency use of CPDGn=26), non-Hodgkin's lymphoma , osteosarcoma (n=12), brain tumours (n=3), Hodgkin's lymphoma (n=2) and pleural mesothelioma (n=1). The patient's characteristics and the different MTX regimens are listed in μmol l−1, all patients had an age-dependent normal value of CreaS before treatment . All patients received intravenous hydration and alkalinisation before, during and after the MTX infusion.A total of 82 patients from institutions in Europe were enrolled in this compassionate and emergency use protocol between May 1997 and March 2002. Complete or nearly complete data were available from 65 patients. For 17 patients, no data are available despite repeated telephone and written efforts. The evaluable 65 patients aged 0.9–71.8 years (median 15.4 years) suffered from acute lymphoblastic leukaemia (μM (median 18.0 μM) (n=65). Two patients were included in the study with MTX levels of 0.52 μM at 94 h (case 1) and 1 μM at 142 h (case 2) after start of MTX and a serum creatinine of six-fold of the initial value. A total of 12 patients had no documented increase of serum creatinine at the time of contact in contrast to the inclusion criteria. Despite the absent renal failure, the principal investigator (UB) decided to give CPDG2 due to very high MTX levels at a late time of contact.Contact with the study centres was established by telephone at a median of 47.5 h (range 19–142 h) after start of the MTX infusion. The MTX levels at the time of contact were between 0.52 and 1082 2 administration are documented in Methotrexate levels and serum creatinine at the first contact and before CPDG2 administration, a median MTX serum level of 11.93 μM (range 0.52–901 μM) was documented (n=58).Immediately before CPDG2 per kg body weight was intended. A vial CPDG2 contains 1000 U dry frozen. In all, 57 of 58 evaluable patients received a dose of between 33 and 60 U CPDG2 per kg body weight (median 50 U kg−1). One patient received only a dose of 17 U CPDG2 kg−1, although the correct amount was available. Carboxypeptidase G2 was administered at a median of 52 h (range 25–178 h) following the start of an MTX infusion of 1.5–12 g m−2 over 4–36 h and was well tolerated. At 15 min after CPDG2 administration, the MTX serum level of these patients was reduced by around 87%.A single dose of 50 U CPDG2. One patient received an additional third dose of CPDG2. Six of these nine patients suffered from an osteosarcoma and received an MTX therapy of 12 g m−2 over 4 h. An additional three patients suffered from ALL and NHL and received MTX infusion of 5 g m−2 24 h−1 (n=2) or 8 g m−2 6 h−1 (n=1), respectively. The median time point of the first CPDG2 dose in these patients was 37.5 h after the start of MTX infusions. The second dose was given at a median of 6 h (range 1.5–90 h) after the first dose. One patient received a second dose of CPDG2 1.5 h after the first dose of CPDG2 due to logistics.Nine patients received a second dose of CPDG2, side effects described by two patients included flushing (n=2) and shaking (n=1). Both had stable vital signs and the symptoms resolved completely without any intervention.After the administration of CPDG2 resulted in a rapid reduction of serum MTX concentrations in all patients. The MTX concentration determined with immunological MTX detection such as TDX or EMIT showed a median decrease of the MTX level of about 87% (range 70–99%) 15 min after CPDG2 administration. Subsequent samples also showed an ongoing decrease of MTX concentrations. Additionally, the MTX concentrations after CPDG2 administration were determined by the HPLC method. The MTX levels estimated by HPLC 15 min after the first CPDG2 injection decreased by a median of 97% (range 73–99%)  . Almost 2, because their serum MTX concentrations determined by immunological methods remained higher than 1 μM after the first dose of CPDG2. The first dose of CPDG2 resulted in a median decrease of 88% (2 did not result in a significant further decline in the serum MTX concentration. Five patients with an MTX level at a median of 8.9 μM (range 3.1–77.6 μM) before the second CPDG2 injection showed a second decrease at a median of 26% (range 5–70%). An additional two patients showed an increase after second CPDG2 injection of 2 and 26%. In one patient, the MTX level after the second dose of CPDG2 is not known and in one case the time between the first and second injections was only 1.5 h and there was no MTX level within the documented level. All patients who received a second dose showed a clearly limited kidney function with median CreaS of 274.4 μmol l−1 (range 168.2–654.9 μmol l−1) before the second administration.Nine patients received an additional dose of CPDGe of 88% . The secIn all, 50% of the patients had a reduced Karnowsky index (⩽60%) due to the MTX intoxication. Gastrointestinal complaints with nausea and diarrhoea were frequent. Severe stomatitis  occurred in 33% of the patients. A total of 66% of the patients showed an increase of serum creatinine of more than 1.5 times of the initial value before MTX therapy. Increased liver values based on increased GPT/GOT were documented in 58% of the patients with CTC scale ⩾2. Only a small number of patients suffered high-grade toxicity of lung and heart/circulation. In five and six patients, respectively, a mild peripheral and/or central neurotoxicity was observed. Two patients suffered from severe central neurotoxic side effects, a female patient additionally from severe peripheral neurotoxic side effects . Further data are listed in 2 insufficiently. Thus, progressive renal failure and fatal septic complications were observed. In an additional patient (case 3), the MTX concentration was lowered by CPDG2 adequately without progressive renal failure. This patient died due to prolonged myelosuppression and its complications (pulmonary haemorrhage due to pulmonary aspergillosis), which were caused by the MTX intoxication and the progressive underlying disease (T-cell ALL) too.Four patients died due to severe myelosuppression and septic complications 7–22 days following the MTX infusion. Three patients  and marked (>97%) decrease in serum MTX concentration (determined by HPLC).After the initial dose of CPDG2 not resulting in a further marked decrease of the MTX concentration. This could be the result of high DAMPA concentrations inhibiting the hydrolysis of MTX by CPDG2  of the initial values 15 min after CPDG2 injection.In addition, substantial crossreactivity of the MTX antibodies with DAMPA used in immunoassays results in overestimation of serum MTX concentrations. Therefore, the MTX and DAMPA serum concentration after CPDG2 is restricted to the extracellular compartment, and the intracellular MTX concentration is initially unaffected by its use. In time, the changed equilibrium between intracellular and extracellular MTX will result in the efflux of intracellular MTX back into the serum, resulting in a rise of serum MTX levels some hours after CPDG2 administration, but this is a relatively slow process. For this reason, rescue with LV must be continued following the CPDG2 administration.Due to its molecular size, CPDGIn this study, six of 65 patients were treated additionally by haemofiltration and/or haemodialysis. These methods must be repeated continuously or daily, in order to reduce the MTX level to nontoxic concentrations. A further risk of these methods results from the necessary vascular entrance, as well as the risk of bleeding secondary to heparinisation and thrombocytopenia. The effectiveness of these procedures cannot be evaluated from our data. Three patients deceased despite these approaches.2 injection due to severe myelosuppression and septic complications 7–22 days following the MTX infusion. The probable reasons for the lethal complications during a prolonged myelosuppression were (1) very high MTX level with an inadequate decrease of MTX levels after the second CPDG2 injection, (2) delayed CPDG2 injection (143.5 h after the start of MTX infusion), (3) a progressive underlying disease (T-cell ALL) and (4) inadequate treatment of severe anaemia in a patient who was a Jehovah witness and refused treatment.In total, four patients died despite CPDG2 followed by long-term LV is highly effective in rescuing patients at high risk of life-threatening MTX toxicity. Carboxypeptidase G2 is safe and well tolerated. No severe side effects were observed in this study. Administration of CPDG2 is most beneficial for patients with MTX-induced renal failure, especially if it is administered within 48–72 h after the start of MTX infusion.In summary, systemic administration of CPDG2 did not result in a significant further decrease of MTX levels, one initial high dose of 100 U kg−1 CPDG2 for patients with extremely high MTX concentrations  may be more effective. For the patients with moderately high MTX levels and renal failure, the application of 50 U kg−1 seems to be sufficient. Due to the high costs of the drug, it may be discussed whether an application of 25 U kg−1 CPDG2 would not be sufficient to lower the MTX levels to nontoxic concentrations in patients with marginal high MTX concentrations and renal failure.For the future, it might be helpful to differentiate between patients with extremly high MTX concentrations and severel renal failure and patients with moderately high MTX concentration and renal failure. Since additional injections at CPDG2 should always be accompanied by a sufficient hydration, alkalisation and a long-term LV therapy, in order to counterbalance the intracellular MTX. Haemofiltration or haemodialysis might be helpful in oliguric or anuric renal failure. In individual cases with renal failure, severe and lethal courses may occur despite a CPDG2 application, in particular if delayed CPDG2 administration did not prevent overt toxicity.The therapy with CPDG


An analysis of risk factors pre-disposing patients to TEEs under epoetin-β therapy was also performed. A total of 2297 patients are included in the analysis.This meta-analysis of all 12 randomised, controlled studies of epoetin-β was used within its licensed indication (Hb initiation ⩽10 g dl−1) or the EORTC recommended level of 11 g dl−1. An increased risk of TEEs is seen for all Hb-initiation level strata and a detrimental impact on survival is seen when initiating epoetin-β therapy at Hb levels >11 g dl−1. We observe no association between high target Hb levels (⩾13 g dl−1) and an increased risk of mortality, disease progression or TEEs with epoetin-β compared with control.Analyses based on various Hb-initiation levels indicate no detrimental impact on survival  and a favourable impact on disease progression  when epoetin-β therapy has no detrimental impact on survival or tumour progression when initiated at Hb levels up to 11 g dl−1. Furthermore, there is no evidence to suggest that high Hb values achieved during epoetin-β therapy are associated with an increased mortality, disease progression or TEE rate.The results of this analysis indicate that epoetin- Anaemia frequently occurs in patients with cancer either as a result of the underlying malignancy or as a consequence of myelosuppressive chemotherapy or radiotherapy, or due to a combination of both (Erythropoiesis-stimulating agents (ESAs) increase Hb levels and reduce transfusion requirements in patients with cancer   . Studies higher) .β conducted in 2301 patients undergoing cancer therapy (β or control (standard treatment) in terms of overall survival, a favourable trend with respect to the risk of disease progression for patients receiving epoetin-β and a higher risk of thromboembolic events associated with epoetin-β treatment  from 12 controlled epoetin-β trials in cancer patients was assessed.The objectives of this analysis are to evaluate the impact on overall survival, disease progression and TEEs of different Hb intervention and target levels for epoetin-β and control (placebo or standard care) with regard to overall survival, disease progression and TEEs during and up to 28 days after end of therapy with epoetin-β.Data presented in this article are derived from an updated meta-analysis of 12 controlled studies designed to evaluate differences between epoetin-β conducted by the drug sponsor  or Boehringer Mannheim) in patients with cancer undergoing treatment (chemotherapy (seven studies), surgery (two studies), radiotherapy (two studies) or radio-chemotherapy (1 study)). The meta-analysis is based on data derived at the individual patient level. Individual study details are summarised in Eligible studies included all randomised, controlled studies of epoetin-β with respect to anaemia correction, in the majority there was no follow-up for survival or tumour progression beyond study treatment plus a standard 28-day period used to assess SAEs, deaths and disease progression. Furthermore, tumour status was not prospectively assessed in many of the earlier trials with short-term follow-up and details of disease progression were routinely reported as adverse events. For the meta-analysis, this information was analysed retrospectively by reviewers blinded to treatment assignment. Four of the studies, which did evaluate the effects of epoetin-β on survival and/or disease progression  by Hb intervention level (baseline Hb level), and (2) by maximum Hb level achieved up to 28 days after end of treatment (target Hb level).Two sets of analyses were performed. One set of analyses included individual patient data from all 12 studies. For these analyses, patients without events were censored at 4 weeks after the last entry in the administration record. A second set of analyses using only pooled patient data from the studies with long-term follow up in which all events were included in the analysis was performed for overall survival  were included in the analysis; four patients were excluded because of not receiving treatment of any kind during the trials. In the studies, three patients in the epoetin group received no epoetin-β and five patients randomised to control received epoetin-β. Patients were analysed according to the treatment received.The 12 randomized, controlled trials enrolled a total of 2301 patients of whom 2297 . Mean baseline Hb level was 10.6 g dl−1 in the control arm and 10.5 g dl−1 in the epoetin-β arm. During treatment, mean maximum Hb level was 13.4 g dl−1 in the epoetin-β arm and 12.0 g dl−1 in the control arm. The mean baseline adjusted Hb area under the curve was 1.24 g dl−1 with epoetin-β compared with 0.07 g dl−1 with control.Median initial weekly epoetin-β (median 3.9 months) and control (median 3.8 months) treatment groups (patients without events from the four studies with long-term follow up were censored 4 weeks after last entry in the administration record). In the four studies with long-term follow-up data, when all events were included, median follow up was also comparable (28.8 months with epoetin-β and 29.8 months with control) (Duration of follow up across the 12 studies was comparable in the epoetin-β or control (standard treatment) in terms of overall survival in the unstratified pooled analysis of all 12 controlled studies . Comparable results were found in the unstratified pooled analysis of the four studies with long-term follow up .As previously described in −1 showed a HR of 0.99      and 2A a N=1426) . These r; N=865) .β treatment on overall survival in patients whose baseline haemoglobin was ⩽10 g dl−1  or ⩽11 g dl−1 , which was consistent with the unstratified analysis . In patients whose baseline haemoglobin levels were above 11 g dl−1, however, the risk of mortality appeared to increase .Comparable results were found in the pooled analysis of four studies with long-term follow up . These dβ and control groups were seen in the number of patients with disease progression .In the unstratified pooled analysis of 12 controlled studies, no significant differences between the epoetin-−1 had a relative risk reduction for disease progression of 27%   with epoetin-β compared with control    and 2B a control . These r0; 1.28) .β over control with respect to disease progression in the subgroups with a baseline Hb level ⩽10 g dl−1  and a baseline Hb level ⩽ 11 g dl−1  with an overall HR for time to TEE of 1.62   when compared with the unstratified analyses . Howeveanalyses .−1 . However, for subgroups with maximum-achieved Hb levels ⩾12 g dl−1 (HR range 1.50–1.59) there was no indication for a further increased risk of mortality.In the pooled analyses of studies with long-term follow up , a shift−1. However, there was no indication of an increased risk in the subgroup of patients achieving maximum Hb levels >13 g dl−1  in which an increased risk of mortality was only seen in the subgroup of patients initiating epoetin-β therapy with a baseline Hb >11 g dl−1.When stratified by baseline Hb level, no significantly increased negative effects on mortality were seen for the epoetin-β, which recommends a Hb-initiation level of ⩽10 g dl−1 and the updated EORTC treatment guidelines  an increased risk of death was seen in the ESA group.Comparable results have been reported by the Cochrane Group from an aggregated study data-based meta-analysis of ESA studies . Analyses of the overall pooled population stratified by baseline Hb level showed similar favourable effects of epoetin-β vs control on disease progression in subgroups with a baseline Hb level of ⩽10 g dl−1, thus supporting the current label, and subgroups with a baseline Hb level ⩽11 g dl−1, consistent with current EORTC guidelines  and an increased risk of thromboembolic events with epoetin-β. These results are consistent with those reported in both meta-analyses of the Cochrane Collaboration . Furthermore, the combination of two or more of these risk factors in epoetin-β-treated patients markedly increased the risk of a TEE. Recently, an association between RBC and platelet transfusions and an increased risk of TEEs and mortality in cancer patients has also been suggested  or within the updated EORTC guidelines , there was no evidence of an increased risk of mortality or disease progression associated with epoetin-β therapy. Consistent with other published meta-analyses, the thromboembolic event rate was significantly increased. The number and combination of pre-disposing risk factors for TEEs at baseline should be taken into account before initiating epoetin-β therapy, and potential preventative measures should be considered. Although there is a clear trend towards a higher risk of overall mortality, disease progression and thromboembolic event rate was observed with increasing baseline Hb values, such a trend was not seen in patients who achieved higher Hb values, especially when Hb values above 13 g dl−1 were exceeded during therapy.This individual patient data-based meta-analysis of 12 controlled epoetin-


Human peripheral blood mononuclear cells (PBMCs) and purified natural killer (NK) cells were used as effectors. The in vitro assays demonstrated hPR1A3 CEA-specific binding and antibody-dependent and CEA-specific killing of human colorectal cancer cell lines by human PBMCs. The effect increased with increasing concentration of antibody and surface CEA, and was lost by using the parent murine IgG1 PR1A3. Killing was also blocked by antibody to the Fc-γIIIA receptor. Purified human NK cells were effective at much lower effector:target ratios than unfractionated PBMCs, indicating that NK cells were the main mediators of hPR1A3-based CEA-specific killing. The results support the development of hPR1A3 for therapy of colorectal cancer.The distribution of carcinoembryonic antigen (CEA) in colorectal cancer (CRC) differs from that in normal colorectal tissue, being found on all borders of the cell membrane and hence enabling access to intravenous antibody, making CEA a good target for antibody-based therapy. The distinctive anti-CEA antibody, PR1A3, binds only membrane-bound CEA. Humanised PR1A3 (hPR1A3) was assessed both  There has been a recent resurgence of interest in the use of monoclonal antibodies for the treatment of cancer, mostly in the setting of metastatic disease, and already a number of antibodies have been licensed for use (for review see Carcinoembryonic antigen (CEA or CEACAM5) was first recognised as a potential human tumour-specific antigen in the 1960s . CEA hasPR1A3 was shown to target the B3 domain and GPI anchor of the CEA molecule by γreceptor are thought to be the main cell type that could mediate ADCC of tumours (Strong evidence for the suggestion that the antitumour effects of antibodies are mediated mainly by ADCC (antibody-dependent cellular cytotoxicity) comes from mouse knockout studies which showed that antibody antitumour effects were largely absent in mice lacking the appropriate Fc receptor  and 1% penicillin/streptomycin. They were incubated at 37°C in a humidified 10% CO2 environment. For the chromium-release and EuTDA assays the cells were suspended in 2% RPMI-1640 medium with 1% glutamine and 10% FCS.All cell lines were maintained in culture in Dulbecco's modified Eagle's (E4) medium with 1% M ethylenediaminetetraacetic acid. Fresh whole blood was mixed with an equal volume of an RPMI-1640/citrate solution  as an anticoagulant.Peripheral blood mononuclear cells (PBMCs) were isolated either from fresh whole blood from healthy laboratory volunteers, having taken informed consent, or from leucodepletion filters obtained from single donors following blood donation . White cells were eluted from the filter using 5 mg (g for 10 min to remove platelets. The resulting PBMCs were resuspended in RPMI-1640/10% FCS/1% glutamine (complete RPMI-1640), kept at room temperature and used within 12 h of preparation.Both sources of PBMC were then processed in an identical fashion. Following Ficoll/hypaque density centrifugation, the PBMC layer was withdrawn from the interface and washed once with RPMI-1640 to remove excess Ficoll, spinning at 800 g  and thenκ monoclonal antibody to CEA . F(ab’)2 derived from the 3G8 clone was obtained from Ancell Corp, Bayport, MN, USA.Two different clones of this antibody were used: MEM154 (Biovendor Laboratory Medicine Inc.) and 3G8 . Both are murine monoclonal IgG1Murine monoclonal antibody 107-1A4 to PSMA was kindly provided by Robert Vessella .This was obtained from DAKO A/S, Denmark, and was diluted to 1 : 100 in RPMI-1640/1% FCS.μg ml−1 in RPMI-1640/1% FCS.clone 4C7 , was obtThis was obtained from the Monoclonal Antibody Service, Clare Hall, Cancer Research UK, London, UK . 2 × 106 target cells were centrifuged at 400 g for 5 min, decanted and labelled by resuspending the pellet in 0.1 ml of 7.4MBq of Na251CrO4. This cell suspension was incubated at 37°C for 60–100 min depending on the optimal labelling time for the particular cell line. Optimal labelling time had previously been determined by choosing the labelling time with the highest maximal lysis:background radioactivity ratio. The cells were then washed twice with RPMI-1640 and suspended in complete RPMI-1640 at a concentration of 1 × 105 cells per ml. Target cells (100 μl (1 × 104)) and PBMCs (100 μl (1 × 106)) were added to microcentrifuge tubes to give an effector: target ratio of 100 : 1. Various antibody concentrations (20 μl) were then added to the relevant tubes. Triton (120 μl of 5%) was added to 100 μl of target cells to obtain maximum release values. All tubes were made up to the same volume using complete RPMI-1640. The tubes were spun at 200 g for 2 min and the pellet of combined target and effector cells was incubated at 37°C for 4 h in the presence of antibody. The tubes were then spun at 200 g for 5 min and 35 μl of the supernatant added to 100 ul Optiphase Supermix  in a 96-well plate. The 51Cr concentration in each well was then determined using a Microbeta plate reader.Peripheral blood mononuclear cells were prepared as above and suspended at a concentration of 1 × 106 target cells suspended in complete RPMI-1640 and incubated for 10–25 min at 37°C depending on the optimal labeling time for the particular cell line, determined as described for the 51Cr-release assays. The relative concentrations of target cells and antibody in the microcentrifuge tubes were similar to those used for the 51Cr release assay. However, varying effector:target cell ratios were used for the fluorescence-based assay. After spinning at 200 g for 2 min the tubes were incubated at 37°C for 2 h and then spun again at 200 g for 5 min. A total of 20 μl of the resulting supernatant was added to 200 μl of Europium in black 96-well plates and the resulting fluorescence was then read in a time-resolved fluorometer using an excitation wavelength of 340 nm and an emission wavelength of 615 nm.Five microlitres of BATDA  at a concentration of 500 U ml−1 in 100 mM TRIS/100 mM MgCl2/100 mM 2-mercaptoethanol with 300 μg ml−1 of 4C7 and incubating this complex at 4°C overnight. The complex so formed was then added at a dilution of 1 : 750 in RPMI-1640/1% FCS. The GAG complex binds to free antigen-binding sites on the rabbit anti-mouse IgG antibody, which is already bound to the PR1A3 attached to the CEA on the cells. The substrate 4-methylumbelliferyl-B-D-galactoside  was first prepared at a concentration of approximately 0.3 mg ml−1 in a buffer of 1 mM MgCl2/100 mM 2-mercaptoethanol . After stirring for 30 min, the solution was filtered to remove excess substrate. This substrate solution was added to each well to start the reaction. After incubation for 40 min in the dark at room temperature, fluorescence was measured using an excitation wavelength of 365 nm and an emission wavelength of 445 nm.CEA levels were determined using a se ELISA . Cells w6 ml−1 and the cells washed once with phosphate-buffered saline (PBSA) containing 2% FCS and then centrifuged at 400 g for five minutes. The resulting pellet was resuspended in cold PBSA containing 2% FCS and incubated on ice with either 100 μl of hPR1A3 at 20 μg ml−1, 100 μl of AUA-1 at 15 μg ml−1 or a medium control for 30 min. The cells were next washed again, with PBSA containing 2% FCS and incubated in a water bath at 37°C for 0, 1, 2 or 3 h. The cells were then incubated with a 1 : 50 dilution of FITC-conjugated anti-human-IgG for hPR1A3 and a 1 : 100 dilution of FITC-conjugated anti-murine IgG for AUA-1 on ice in the dark for 30 min before being washed again and resuspended in PBSA containing 2% FCS. The cells were then passed through a fluorescence-activated cell sorting (FACS) Calibur flow cytometer and the results analysed using CellQuest software.The concentration of cells to be analysed was adjusted to 1 × 10μl per 107 cells) which contained antibodies against T cells, B cells, stem cells, dendritic cells, monocytes, granulocytes and erythroid cells. Following incubation for 10 min at 4°C, the NK Cell magnetic Microbead Cocktail was added. This was left for 15 min at 4°C and the cells then washed with MACS buffer (PBSA/0.5% FCS/2 mM EDTA) and centrifuged. The cell pellet was resupended in 0.5 mls MACS buffer and then passed through an LS magnetic column to remove cells that bound the antibodies in the cocktail. The cells that passed through were then collected and suspended in RPMI-1640 with and without IL-2 (Peprotech) at 10 ng ml−1 and incubated overnight at 37°C. Aliquots pre and post sorting were taken for FACS analysis with the antibodies CD3, CD56 and CD16 and the appropriate isotype controls. Thus, 1 × 106 cells were suspended in 2 ml of FACS buffer (PBSA/1%FCS/1% sodium azide) and centrifuged. The supernatant was removed and the cells resuspended in the residual buffer. The antibodies were then added and left for 20 min at 4°C before washing and then fixing with 300 μl of 2% paraformaldehyde. The fixed cells were analysed on a FACS Calibur flow cytometer. Gating of the lymphocyte population in the forward versus side scatter plot revealed a purity of at least 85% with respect to CD16 and CD56 binding. The resulting NK cells were used in cytotoxicity assays with hPR1A3 against SKCO-1, which is known to express CEA.Fresh PBMC or eluted PBMC from leucodepletion filters were enriched for NK cells using a Human NK Cell isolation kit (Miltenyi Biotec) following the manufacturer's instructions. This involved adding the NK cell Biotin-Antibody cocktail  that were isolated from PBMC from a healthy volunteer. CEA was added to achieve final concentrations of 2 and10 μg ml−1 was added to hPR1A3 . The mixture was incubated in a 1.7 ml eppendorf microtube for 45 min at room temperature. Humanised PR1A3 alone, at a concentration of 20 μg ml−1, and medium alone were similarly incubated in a microtube. Cells to be assayed with these various mixtures were adjusted to a concentration of 1 × 106 ml−1 in RPMI-1640 complete medium and washed with FACS buffer (PBSA/1%FCS/1% sodium azide). The resulting pellet was resuspended in 10 μl FACS buffer and then incubated on ice with 100 μl 20 μg ml−1 mPR1A3, hPR1A3/CEA (as prepared above) or medium control for 30 min. The cells were then washed twice with FACS buffer and incubated on ice in the dark for 30 min with a 1 : 50 dilution of FITC-conjugated anti-human-IgG (for hPR1A3). The resulting labelled cells were washed again with 2 ml of FACS buffer and centrifuged at 400 g. The supernatant was removed completely and the cells resuspended in 300 μl of 2% paraformadehyde in PBSA. The labelled cells were analysed using a FACS Calibur flow cytometer as described before.Soluble CEA at a final concentration of 10 Percentage lysis of the cell lines in the cytotoxicity assays was calculated as /(maximum release−background release) × 100. Percentage specific lysis was calculated as /(maximum release−antibody independent release) × 100. The s.e.m. of multiple experiments was calculated using Graphpad Prism software, San Diego, CA, USA. Standard normal distribution tests were used to assess the significance of the differences found.Humanised PR1A3 showed specific binding to MKN45, a high CEA-expressor. After three hours incubation of MKN45 at 37°C with hPR1A3, no change was observed in the amount of antibody detected on the cell surface . This is51Cr using different antibody concentrations. In the absence of antibody, PBMCs effected a low but significant level of spontaneous killing. The level of killing increased with increasing concentrations of antibody, as would be expected for ADCC  receptor To establish that the major killing effect seen with hPR1A3 was actually due to NK cells as conventionally defined, purified NK cells were used in assays of hPR1A3 killing of the high CEA expressing colorectal cell line, SKCO-1. NK cells were enriched from PBMCs obtained from buffy coat (NBS) using the Human NK Isolation kit, as described in Materials and Methods.The extent of enrichment for NK cells is illustrated by the FACS analysis shown in μg ml−1) on its own, or after preincubation with soluble CEA (10 μg ml−1) was investigated using a FACS analysis. The results, illustrated in The binding to the high expressing cell line SKCO-1 of hPR1A3 2 fragment) also abolishes antibody independent killing may be explained either by antibody binding to CD16-bearing cells reducing their mobility in the wells due to formation of clusters of CD16+ cells or that killer cells may destroy CD16+ cells which have bound the antibody.Human NK cells are known to express CD16 (homologous to the Fc in mice ) and areThe affinity of hPR1A3 for CEA appears to be relatively low as reflected by the higher concentrations of PR1A3 needed to mediate ADCC as compared to cetuximab see . Howeverin vivo situation, including especially the development of an immune response against the toxins or enzymes linked to a therapeutic antibody.Much research is now directed at conjugating antibodies with radiolabels or toxins. Although this has had some success in experimental animal models, there remain significant problems in the true in vitro.We suggest that the appropriateness of CEA as a therapeutic target, together with our evaluation of antibody hPR1A3's mediated ADCC activity makes this antibody a very attractive target for clinical development as a naked antibody. The main challenge may be to enhance PR1A3's ADCC activity, and this may be achieved by glycoengineering its Fc hinge region (As previously discussed, only a small percentage of antibody administered intravenously actually reaches the cells of a solid tumour ((


SDF1 gene variation, plasma SDF-1alpha level, and the number and function of circulating EPCs. We also tested whether EPC number and function was related to CXCR4 gene variation.Stromal cell-derived factor-1 (SDF1) and its receptor CXC chemokine receptor 4 (CXCR4) play a critical role in progenitor cell homing, mobilization and differentiation. It would be interesting to assess the predictive value of SDF-1alpha level for EPC number, and to ascertain whether there is a relationship between SDF1 and CXCR4 genes, and measured blood SDF1α level as well as EPC number and function. SDF1α levels were correlated with age, gender, alcohol consumption, circulating reticulocyte numbers, and concentrations of matrix metalloproteinase-9, C-reactive protein, cystatin C, fibrinogen and homocytein. In blood samples taken in 2005, EPC number was inversely associated with SDF1α level (p<0.001). EPC number in 2005 was also inversely associated with SDF1α level in 2000 (p = 0.009), suggesting a predictive value of plasma SDF1α level for EPC number. There was an association between the SDF1 gene rs2297630 SNP A/A genotype, increased SDF1α level (p = 0.002) and lower EPC number (p = 0.006).We genotyped a cohort of individuals who participated in the Bruneck Study for single nucleotide polymorphisms (SNPs) in the Our data indicate that a SDF1 gene variation (rs2297630) has an influence on SDF1α level and circulating EPC number, and that plasma SDF1α level is a predictor of EPC number. Asahara and colleagues in 1997 demonstrated for the first time that purified CD34 positive haematopoietic progenitor cells from peripheral blood could differentiate, ex-vivo, into an endothelial phenotype and were named Endothelial Progenitor Cells (EPCs)SDF1 or CXCR4 gene in mice led to early embryonic lethality due to abnormality in the cerebellar and gastrointestinal vasculature and in hematopoiesis developmentSDF1 gene transfer using the adenovirus infection techniqueAnimal studies have indicated that SDF1  and its receptor CXC chemokine receptor 4 (CXCR4) plays a critical role in progenitor cell homing, mobilization and differentiation. Inactivation of the SDF1 gene can have an influence on SDF1α levels+ cell mobilizationSDF1 gene variation, SDF1α level and circulating EPC number. In addition, we tested whether there was also a relationship between variation in the gene encoding the SDF1α receptor CXCR4 and EPC number.There is emerging evidence indicating that variation in the human SDF1 and CXCR4 genes. Blood samples taken in 2000 (n = 684) and 2005 (n = 574) respectively were used for measurements of SDF1α levels. EPC number and function were assessed in blood samples collected in 2005 (n = 571 for EPC number and n = 542 for EPC colonies number respectively). Subjects with and those without EPC number and EPC function assessments did not differ in age, sex and cardiovascular risk factors. The appropriate ethics committees (Autonome Provinz Bozen-Sanitatsbetrieb Bozen Ethikkomittee) approved the study protocol and all study subjects gave their written informed consent before entering the study.The subjects of this study were residents of the Bruneck area in the Bolzano Province of Italy, who participated in the Bruneck StudySystolic and diastolic blood pressure was taken with a standard mercury sphygmomanometer after at least 10 min of rest (mean of three independent measurements). Hypertension was defined as blood pressure ≥140/90 mm Hg or the use of anti-hypertensive drugs. The average number of cigarettes smoked per day was noted for each smoker and ex-smoker. Diabetes was diagnosed according to ADA criteria. Assessment of regular alcohol consumption was performed with a standardized questionnaireand quantified in terms of grams per day (g/day).EPC numbers in the blood samples were determined as described previouslyThe EPC-CFU assay performed as described previouslyCommercially available SDF1α and matrix metalloproteinase-9 (MMP9) ELISA kits  were used to determine plasma SDF1α and MMP9 levels. All ELISA tests were carried out at room temperature on freshly thawed plasma samples. The concentration was determined by comparison with a standard curve, following manufacturer's instruction. Other laboratory parameters and vascular risk factors were all examined by standard methodsSDF1 and CXCR4 genes from the HapMap database (data release in June 2005). Three SNPs in SDF1  and two in CXCR4 (rs16832740 and rs12691874) to capture SNPs with a minor allele frequency >0.2 at these loci and R2>0.8 were typed in this study. In addition, we studied the rs266085 SNP which had been reported to influence SDF1 expressionWe selected tagging SNPs in the eB-transformed. Non-parametric tests yielded very similar findings (data not presented). Associations of EPC number, EPC-CFU and SDF1α level with SDF1 and CXCR4 genotypes were assessed using generalized linear models adjusted for age and sex. Multivariate models additionally included standard cardiovascular risk factors and the factors associated with SDF1α levels . A Bonferroni correction was performed to account for the multiple comparisons (5 SNPs in SDF1 and 2 in CXCR4) performed.The data were analyzed using the SPSS 15.0 and BMDP software packages. Continuous variables were presented as means±SD or medians (interquartile range), and dichotomous variables as percentages. Correlations between EPC number and EPC-CFU, SDF1α level and other parameters were estimated by calculation of crude and partial (age/sex-corrected) Pearson correlation coefficients. Variables with a skewed distribution were logB. Kolmogorov-Smirnov p>0.05). Mean SDF1α levels in 2000 and 2005 were 2652 pg/ml and 2565 pg/ml, with a range of 880 pg/ml to 5595 pg/ml and 1322 pg/ml to 4174 pg/ml, respectively. SDF1α levels in 2000 and 2005 were highly correlated , indicating that the SDF1α level is relatively consistent over time. As expected, both plasma SDF1α level measured in 2000  and 2005  increased with age , indicating that SDF1α has an influence on EPC number in the general population. Importantly, EPC number in 2005 was also associated with SDF1α level in 2000 (p = 0.009), indicating a long-term predictive value of SDF1α level for circulating EPC number (EPC number and EPC-CFU number (per 1 ml blood) displayed a non-normal distribution, with the majority of subjects (more than 75%) having 0 to 1000 EPC and 0 to 340 EPC-CFU, respectively. SDF1α levels were significantly and inversely associated with EPC number (p<0.001) in samples collected in 2005 1C, indSDF1 and CXCR4 SNPs examined in this study are shown in . The genotype distributions were consistent with Hardy-Weinberg equilibrium.Allele and genotype frequencies of the SDF1 rs2297630 SNP was associated with plasma SDF1α level (p = 0.002) and circulating EPC number (p = 0.006), with the A/A genotype associating with higher SDF1α level and lower EPC number  and the relationships of the SDF1α levels at these different time points with circulating EPC numbers. A key finding from our study is the high degree of correlation the two SDF1α measurements in blood samples taken in 2000 and 2005 respectively, suggesting that although SDF1α levels are correlated with cardiovascular risk factors, the levels in an individual are relatively consistent over time. In addition, the study revealed an association between SDF1α level in 2000 and EPC number in 2005, suggesting a long-term predictive value of SDF1α level for EPC number. Another important, novel finding of this study is that there is an association between variation in the + cells in peripheral blood was lower but the level of SDF1α was much higher at 14 days after ischemia, as compared with control miceSDF1 is a member of the chemokine CXC subfamily originally isolated from murine bone marrow stromal cells and is expressed also by stromal cells of various tissuesSDF1 gene. In particular, we found that SDF1α level and circulating EPC number are associated with the SDF1 gene rs2297630 SNP in the Bruneck study cohort. A potential interpretation for this novel finding is that variation in the SDF1 gene can influence EPC number via an effect on the level of SDF1α. The rs2297630 SNP is located in intron 3 of the SDF1 gene. It is possible that the association of this SNP with SDF1α level and circulating EPC number has arisen from a direct functional effect of this SNP on SDF1 expression or mRNA splicing. Alternatively, the rs2297630 SNP might be a functionally neutral marker that is in linkage disequilibrium with a functional polymorphism located elsewhere at the SDF1 locus.A recent study showed that there is significant correlation in the number of circulating EPCs between parents and their offsprings, leading to the hypothesis that EPC number is, at least in part, genetically regulatedSDF1 SNPs, rs266085 and rs1801157, have been reported to be associated with SDF1 levels+ cell mobilizationSDF1 locus due to linkage disequilibrium. This possibility is consistent with the fact that the SDF1 gene is transcribed into two isoforms, SDF1α and SDF1β, and that the rs1801157 SNP is located in the 3′ untranslated region of the SDF1β transcript but not in the SDF1α transcriptTwo other Some particular attentions should be drawn regarding our findings. Firstly, the consideration of the nature of EPC is important, since there is no a unique criteria for identification of EPC, yet. Immense attention regarding the definition of EPC has recently been drawn due to the fact that the characterization and function of EPC isolated with different methodologies were quite different from each otherIn summary, our data indicate that although the SDF1 level and EPC number may both increase in response to acute ischaemic events, the relationship between blood SDF1 level and EPC number is complex and they are inversely correlated in the normal situation as observed in the Bruneck study cohort which was recruited from the general population. Our data also indicate that circulating EPC number is influenced by variation in the SDF1 gene likely via an effect of the genetic variation on SDF1α expression. These findings help understand the mechanisms underlying the inter-individual variability in EPC number which has an implication in the pathogenesis of atherosclerosis and other cardiovascular diseases.


A maximum entropy approach is proposed to predict the cytotoxic effects of a panel of colchicine derivatives in several human cancer cell lines. Data was obtained from cytotoxicity assays performed with 21 drug molecules from the same family of colchicine compounds and correlate these results with independent tubulin isoform expression measurements for several cancer cell lines. The maximum entropy method is then used in conjunction with computed relative binding energy values for each of the drug molecules against tubulin isotypes to which these compounds bind with different affinities.αβI and αβIII tubulin isoforms are the most important isoforms in establishing predictive response of cancer cell sensitivity to colchicine derivatives. However, since αβI tubulin is widely distributed in the human body, targeting it would lead to severe adverse side effects. Consequently, we have identified tubulin isotype αβIII as the most important molecular target for inhibition of microtubule polymerization and hence cancer cell cytotoxicity. Tubulin isotypes αβI and αβII are concluded to be secondary targets.We have found by using our analysis that αβIII as a target for optimized chemotherapy drug design.The benefit of being able to correlate expression levels of specific tubulin isotypes and the resultant cell death effect is that it will enable us to better understand the origin of drug resistance and hence design optimal structures for the elimination of cancer cells. The conclusion of the study described herein identifies tubulin isotype  To estimate ICItop is the response obtained at very low/no drug concentration and X denotes dose in logarithm unit. After regression, parameters, Itop, Ibot and log IC50 were normalized to Itop . Ibot is a measure of the maximum effect of the drug. A Monte Carlo simulation [50 of cell lines exposed to twenty colchicine derivatives are listed in Table for regression, where HILL is a measure of the steepness of the transition region and was fixed at a value of 2.5. mulation  was applFollowing cytotoxicity assays which gave us an indication that the designed colchicine derivatives have led to encouraging outcomes for a panel of diverse cancer cell lines, our next task was to determine the expression levels of tubulin isotypes before and after exposure of the cells to the toxic agents including colchicines and D20 at the estimated 25% of lethal concentration of dose. This type of experiment would provide information regarding the regulation by the cells of the target protein, tubulin, and its specific isoforms.μg/ml DNase and 100 μg/ml RNase. Protein was quantified using Micro BCA protein assay kit . An equal amount of protein (20 μg) was boiled in SDS loading buffer containing 0.1 M DTT and resolved by electrophoresis in 7.5% SDS polyacrylamide gels . Biotinylated protein ladder  and Kaleidoscope pre-stained protein standard  were included for assessment of molecular mass of target proteins. The gels were transblotted to Hybond-C nitrocellulose membranes  and the membranes were blocked in 5% ECL Advance blocking solution  for 1 h at room temperature. Immunodetection was performed by incubating the membrane overnight at 4°C with murine monoclonal antibodies to β tubulin isotypes I through IV with murine monoclonal antibodies to β tubulin isotypes I through IV or with a murine monoclonal antibody to β-actin as a housekeeping gene product control  followed by incubation with the secondary antibody, anti-mouse IgG HRP conjugate . Antibody dilutions were as follows: βI , βII , βIII , and βIV . The secondary antibody solution also included 1:10,000 anti-biotin HRP-linked antibody to visualize the protein ladder. All antibodies were diluted with 2.5% ECL Advance blocking solution. Target proteins were visualized by enhanced chemiluminescence with ECL Advance Western Blot detection kit and captured on Hyperfilm-ECL film (both from Amersham Biosciences). Image analysis was performed with a personal densitometer (Amersham Biosciences). Relative quantities of target proteins were determined using ImageQuant and normalized to β-actin levels in each sample. Furthermore, we normalized it across four isotypes. The data are shown in Table Briefly, frozen cell pellets from six human cell lines A549, MCF7, CEM, HeLa, M006X, M010B were thawed on ice and homogenized on ice in 10 mM Tris pH 7.4 and 0.3% SDS with 1% protease and phosphatase inhibitors  and centrifuged at 12,000 × g at 4°C. The supernatants were treated with 50 50 values from cytotoxic measurements on the cell lines exposed to these drugs, what are most likely expression levels of specific tubulin isotypes?iven the information regarding the binding free energy between individual tubulin isotypes and colchicine derivatives and the IC ".Our goal was to utilize information such as binding free energy values between a toxic agent and a molecular target in order to estimate tubulin isotype expression levels present in cytotoxcity assays. This is exactly the type of question that the method of maximum entropy is designed to answer -34. Speca priori chosen function each time additional information is acquired. Note that the probability distribution represents our state of knowledge of the systems in a specific state. Suppose the system of interest is characterized by label i and the variable Hi represents some properties of the system in state i. For example, the label i may represent a binding mode of target proteins and ligands. Furthermore, suppose that probability values μi are given as priors before any measurements are performed. Suppose also the new information H in the form ofFollowing Jaynes' method of maximum entropy (MaxEnt) , which iPi that codifies this new information H and the prior is μi determined by maximizing the relative entropy given byis acquired. The preferred subject to the normalization constraint and the constraint in Eq. (2). The preferred posterior probability is then given by,β can be determined by substituting Eq. (4) back into Eq. (2).where the partition function is defined in a standard way as The method of ME provides a robust and universal route for information processing with the least bias. The ME method ensures that the ME posterior probability distribution is preferred over any other probability distributions used to characterize the system of interest as long as the information used to update from a prior is relevant to the system. Thus, to estimate the expression levels of tubulin isotypes in cytotoxicity assays involving the novel colchicine derivatives, we only need to focus on determining the information that is relevant to the interactions between tubulin isotypes and colchicine derivatives and the relationship with the outcomes of the cytotoxicity assays.Gi with specific weighting factors Pi representing relative expression levels of individual isotypes,The interaction between tubulin isotypes and colchicine derivatives (colchicine is denoted by C and its derivatives are collectively denoted by C') is best described by the binding free energy of tubulin isotypes and either C or C'. The total binding free energy in each cell line is the average of binding free energy of M tubulin isotypes ΔG⟩ if can be measured, then the preferred weighting or the expression level can be determined.where subscript i labels the type of tubulin isotype. Based on the ME method, ⟨Δ50. The studies of Tian and Haffner show a linear relation between cytotoxicity-derived values of logIC50 and the binding free energy [Although it is difficult to directly measure the binding free energy for the entire cell line, the information regarding the interaction between tubulin isotypes and colchicine derivatives can still be described by the cytotoxicity measurement of the values of log ICe energy ,(6)50 as clearly seen from Eq. (6), we still require the individual contribution values towards the total binding free energy coming from tubulin isotypes ΔGi for each of the administered colchicine derivatives. Because of the complicated conformational changes in the physical binding processes, calculations of the binding free energy of tubulin isotypes i and colchicine 50 values obtained from cytotoxicity measurements.Even though the total binding free energy can be estimated from log ICG⟩ from log IC50 using ΔGi estimates from ΔΔGibind_, we can now re-formulate the constraint equation that relates the cytotoxicity of colchicine derivatives given by the values of log IC50 and the binding free energy between tubulin isotypes and colchicine derivatives asFinally, having estimated ⟨Δα = C or C' to denote the cell line exposed to colchicine or colchicine derivatives, respectively.where we use superscript μi with the information given by Eq. (7) asThe ME method gives the probability distribution that is updated from a prior βα is determined from Eq. (7). This will be used as the normalized expression level of tubulin isotype i in the cell line exposed to colchicine derivative α. However, because where the partition function gi is a dummy variable and we set μi = 1 to indicate that no other prior information is included. Furthermore, the constraint, Eq. (7) can be rewritten aswhere Therefore, the binding free energy involving a tubulin isotype and colchicine can be estimated from μi. Furthermore, the practically of Eq. (11) requires an initial guess of βC' in order to solve Eq. (7). In our studies, in order to generate a statistically relevant data set, we will repeat the same calculation one hundred times with various initial guesses for βC' generated by random selection. Both the mean value βC' and its standard deviation will be used in our calculations.where we consider the normalized expression level αβIIa and αβIIb are structurally indistinguishable in some experiments, as are the isotypes αβIVa and αβIVb, we average relative binding energy of isotypes αβIIa and αβIIb and isotypes αβIVa and αβIVb as isotypes αβII and αβIV, respectively, to account for this property.To validate the proposed approach, we consider the cell lines used except M006X and one colchicine derivative D20 as the benchmark because of the sufficient experimental expression data for these five cell lines exposed to D20 . The results of our analysis are plotted in figure 50 values. The remaining labels show the same order of tubulin isotype expression. The figure shows several important features. First, tubulin isotype αβIII is likely to show the highest expression level for the cell line A549, Hela, M010B and M006X exposed to the colchicine derivatives D03, D04, D05, and D20. For MCF-7, it has the highest expression level for the colchicine derivatives D03, D04, and D06. Second, for colchicine derivatives D09 and D13, Figure αβI has the highest expression level in all cell lines except MCF-7 and CEM. Furthermore, it also has the highest level for cell line CEM with three out of five derivatives. Third, for cell line MCF-7, it is either isotype αβII or αβIII that has the highest expression level. Finally, the frequency score of the isotypes with the highest expression level is 13, 7, 26, 5, 35 and 28 for the tubulin isotypes αβI, αβII, αβIII, αβIV, labelled "U" and "0", respectively. Isotype αβIII is ranked first and is followed by αβI and αβII over all. Fourth, Figure αβIII, the potency is unlikely a factor to influence the binding affinity. Finally, this suggests that tubulin isotypes αβIII followed by αβI and αβII are the first three primary targets of colchicine derivatives in all cell lines except CEM giving us important insights for future optimization of drug design based on colchicine derivatives.We further summarize and illustrate the tubulin isotype distribution with the highest expression levels in cell lines exposed to the twenty colchicine derivatives in figure αβIVa and αβIVb are structurally distinguishableOnly tubulin isotype αβIVa and αβIVb are structurally distinguishable in cell lines. Because experimental measurements on expression levels are only available for the isotype αβIV, we simply assign both αβIVa and IVb with the same prior, which is half of the normalized expression level of isotype αβIV for the calculations. Furthermore, we only take A549 cell line as an example for our investigations , were exposed to colchicine and were exposed to the compound D20. The maximum entropy method is used in conjunction with binding energy calculations for the colchicine derivatives, as they were found to interact specifically with tubulin isotypes. We found that depending on the use of assumptions regarding the priors in the method applied to the probability distribution functions, various outcomes may be obtained. However, in the presence of experimentally available information, a much more consistent picture emerges. Most importantly, in almost all cases studied we have identified the tubulin isotype αβIII, for optimized chemotherapy drug design compared to earlier efforts in this area. By narrowing down the focus of tubulin targets to this isotype, most dramatically regulated by cancer cells when exposed to the colchicine derivatives, both the efficacy and specificity of treatment will hopefully be improved.We have demonstrated the applicability of the maximum entropy approach in predicting cytotoxic effects based on limited information such as the relative binding energy values for the cytotoxic agents used. Namely, given the relative binding free energy of tubulin isotype and colchicine derivatives, the proposed approach predicts the tubulin isotype expression levels in various cell lines exposed to colchicine derivatives. Our studies also provide a better defined molecular target for the action of these anti-mitotic drugs, namely, tubulin isotype αβIII tubulin isotype, hence the best compounds revealed in this study may offer a potential improvement in clinical outcomes. An interesting question to ask is why the currently used compounds show good efficacies without specifically targeting tubulin isotype αβIII. The probable reason why taxanes and vinblastine are successful is that they preferentially interact with the αβII isotype [αβIV, an isotype widespread in normal tissues that is not common in cancers [Unfortunately, the currently used chemotherapy drugs do not particularly target  isotype ,46 which isotype , but has isotype ,48. Not  isotype -51. In c cancers . Hence, αβIII isotype of tubulin singled out in the present paper is an almost perfect target for breast cancer chemotherapy because a) it is found in many tumors, especially those that are metastatic and aggressive, including breast tumors [αβII, occurring largely in the brain and the testes [αβIII accounts for only 25% of brain αβ-tubulin while αβII constitutes 58% [αβII is found in both neurons and glial cells, αβIII occurs only in neurons [The t tumors ,52-55; ae testes . αβIII autes 58%  suggestiutes 58% . Further neurons .αβIII would be an excellent target for anti-tumor drugs [αβIII-specific drug. The seco-taxoid, IDN 5390 [αβIII. This drug is very effective against paclitaxel-resistant cell lines over-expressing αβIII. The epothilone derivative ixabepilone which is very effective against human cancer cell lines and xenograft models that over-express αβIII and under-express αβII, is now recommended for use with metastatic taxane-resistant or taxane-untreated breast cancers [in vitro that either ixabepilone or IDN 5390 binds well to αβIII and poorly to other isotypes. The in vivo data indeed indicate that these compounds target αβIII [αβIII isotype---nor of the targets to be avoided, such as the αβI or αβII isotypes.Other researchers have already realized that or drugs  and haveIDN 5390 ,59 was e cancers ,61. Howeet αβIII  but do net αβIII , and theet αβIII  indicateαβIV isotype, while it binds very poorly to αβIII. These novel compounds were designed to bind well to αβIII and hence should not cause the same type of toxicity as does colchicines.On the other hand, our novel colchicine derivatives bind at a different site on tubulin than do either IDN 5390 or ixabepilone. Thus, we are exploring a new area here. There is no reason to expect these derivatives to have the same toxicity and side effects as does colchicine, the parent compound. Since its toxicity arises from the fact that standard colchicine has a very high affinity for the widespread The authors declare that they have no competing interests.CYT carried out the development of maximum entropy approach, performed tubulin expression level analyses and drafted the manuscript. JM carried out the QM/MM binding affinity calculation. TH was involved in the preparation of tubulin isotype structures for binding affinity calculations and the analysis of binding modes for colchicine derivatives. PW carried out cytotoxicity statistical analysis. LJ performed all cytotoxicity experiments. EI carried out the essential work required for the tubulin isotype expression assays. RFL contributed to the identification of specific tubulin isotypes as chemotherapy targets and participated in tubulin expression assays. JAT conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


The first two amino acids are not necessary since the eptapeptide 206–212 was equipotent in both assays (IC30 of 12.5 μM and 420 μM). Of the two pentapeptides 204–208 and 208–212 only the latter showed inhibitory activity in both models although the potency was much reduced (IC30 of 170 μM and 630 μM) compared with that of the parent nonapeptide. Comparison of peptide 204–212 effects with those of its analogues on LC1 and LC2 indicate that lysine 208 and aspartic acid 211 are essential in order to maintain a fully active nonapeptide.Peptide 204–212 of lipocortin (LC) 5 inhibited porcine pancreatic phospholipase A


In the second sentence of the section "Cell Culture and Transfections" of the Materials and Methods, the concentration of the siRNA solution is incorrect. It should read: "...2 µL of a 1 µM siRNA solution..."


NRH:Quinone Oxidoreductase 2 (NQO2) has been described as having no enzymatic activity with nicotinamide adenine dinucleotide (NADH) or NADPH as electron donating cosubstrates. Mitomycin C (MMC) is both a substrate for and a mechanistic inhibitor of the NQO2 homologue NQO1. NRH:quinone oxidoreductase 2 catalysed the reduction of MMC at pH 5.8 with NADH as a co-factor. This reaction results in species that inhibit the NQO2-mediated metabolism of CB1954. In addition, MMC caused an increase in DNA cross-links in a cell line transfected to overexpress NQO2 to an extent comparable to that observed with an isogenic NQO1-expressing cell line. These data indicate that NQO2 may contribute to the metabolism of MMC to cytotoxic species. Mitomycin c (MMC) is an anticancer antibiotic used in the treatment of superficial bladder cancer and other tumours. As a prodrug, the parent compound is relatively nontoxic compared to the highly electrophilic metabolites formed following sequential or simultaneous two electron reduction of the quinone to the hydroquinone . Many en50 and NQO1 activity in the National Cancer Institute 60 cell line panel presented evidence of a more heterogeneous nature that NQO1 has a role in MMC bio-activation  has recently been described as an enzyme of surprises and mysteries . In thisμm 100 × 4.6 mm HPLC column was purchased from Jones chromatography .hrNQO1, hrNQO2  was evaporated under nitrogen and the residue resuspended in 400 μl water. Isocratic HPLC separation of MMC was carried out on a Waters  Alliance 2695 separation module and 2487 dual wavelength detector with detection at 365 nm. Mobile Phase comprised of 72% 10 mM ammonium formate (pH 6.5) : 28% acetonitrile at a flow rate of 0.3 ml min−1. Injection volume was 5 μl with a run time of 10 min.Reduction of MMC was carried out in a 100 mM and 100 μM MMC. Aliquots were removed at 0, 5, 10, 15, 20 and 30 min for HPLC analysis. Mitomycin C concentrations remaining were interpolated from a standard curve and initial rates were calculated as the initial slope of a second-order curve of the data.NQO2 kinetic studies were carried out as above at pH 5.8 with a range of NADH concentrations of 0.25–4 mM DCIP and ±0.01 mM dicumarol in 25 mM Tris-HCL buffer (pH 7.4). The reaction was initiated by the addition of NADH to a final concentration of 0.2 mM and a final volume of 300 μl. The absorbance at 600 nm was measured at 10 s intervals over 2.5 min at room temperature, monitored using a Spectramax 250 spectrophotometer. All reactions were carried out in 96-well plates and each sample was measured in triplicate. The concentration of oxidised DCIP remaining at each time point was calculated from a molar extinction coefficient of 21 ml μmol−1cm−1. The reaction rate in nanomolar DCIP reduced per min per mg of total protein was calculated from a plot of A600 against time.The NQO1 activity of cell lysis supernatants was determined by an adaptation of a previously published method  : acetonitrile (50 : 50) was injected onto a Genesis C18 120A 4 μm 100 × 4.6 mm HPLC column with a mobile phase of 80% 0.02 M phosphate pH 6 : 20% methanol at an isocratic flow of 1 ml min−1. CB1954 was detected at a wavelength of 325 nM and a retention time of 7 min and the concentration remaining was interpolated from a standard curve. The rate of CB1954 reductase activity was calculated by the initial velocity per mg of protein.CB1954 reduction was assayed as previously described  slide. Once the agarose had set, the slides were irradiated at 10 Gy prior to lysis and subsequent denaturation at pH 13. Cells underwent horizontal electrophoresis at a constant 300 mA and between 15 and 20 V for 20 min. The slides were washed in deionised H2O and fixed in 70% EtOH and allowed to dry overnight. The cells were stained with SYBR green and 3D images acquired by confocal microscopy. Comets were analysed by Komet 5 software DNA alkylation was assessed by modified comet assay as previously described . No reduction of MMC was seen in a solution containing BSA, lactose and PBS salts under the same conditions (μM) (data not shown). No reduction of MMC occurred at any pH with either enzyme in the absence of NADH (data not shown).Either hrNQO1 or hrNQO2 alone catalysed the reduction of 100 nditions . Reductinditions . NRH:quiμM quercetin and partially inhibited by 100 μM dicoumarol when compared to the reaction in the absence of the inhibitor  , when co−1 mg−1. NQO1 was undetectable in the NQO2 transformed cell line (TH7) which had a mean NQO2 activity of 8.2±2.9 nmol min−1 mg−1. Following a 1 h incubation of each cell line in 10 μM MMC at physiological pH and 37°C the mean Olive tail moment was unchanged compared to untreated cells in the F179 cells. In contrast both the NQO1 (hdt) and NQO2 (TH7) cell lines had a decreased Olive tail moment following MMC treatment compared with untreated cells (V79 chinese hamster lung fibroblasts cells transformed to overexpress NQO1 or NQO2 were used to determine the relative degree of DNA damage caused my MMC. Neither NQO1 nor NQO2 activity was detectable in the empty plasmid transformed cell line (F179). NRH:quinone oxidoreductase 2 was undetectable in the NQO1 transformed cell line (hdt) which had a mean NQO1 activity of 1487±29 nmol mined cells .Km for NADH three- to four-fold higher than that of NQO1  purified rat hepatic NQO1 . A further study suggested this activity was due to Mr 58 000 glucose regulatory protein (GRP58)  . There iThe activity described in this study is unlikely to be extrapolated to other quinone substrates of NQO1 given the unique combination of low affinity of NQO1 for MMC and the low affinity of NQO2 for MMC and NADH. Mitomycin C is clearly metabolised by NQO2 in the presence of NADH to reactive species that both inhibit NQO2 and react with DNA to form cross-links. This study offers no suggestion that NQO2 functions physiologically as an enzyme. However, these data indicate that NQO2 may contribute to the bioreductive activation of MMC, particularly in an aerobic environment.