Plasma tumor gene conversions after one cycle abiraterone acetate for metastatic castration-resistant prostate cancer: a biomarker analysis of a multicenter international trial.

BACKGROUND: Plasma tumor DNA fraction is prognostic in metastatic cancers. This could improve risk stratification before commencing a new treatment. We hypothesized that a second sample collected after one cycle of treatment could refine outcome prediction of patients identified as poor prognosis based on plasma DNA collected pre-treatment. PATIENTS AND METHODS: Plasma DNA [128 pre-treatment, 134 cycle 2 day 1 (C2D1), and 49 progression] from 151 chemotherapy-naive metastatic castration-resistant prostate cancer (mCRPC) patients in a phase II study of abiraterone acetate (NCT01867710) were subjected to custom targeted next-generation sequencing covering exons of these genes: TP53, AR, RB1, PTEN, PIK3CA, BRCA1, BRCA2, ATM, CDK12, CHEK2, FANCA HDAC2 and PALB2. We also captured 1500 pan-genome regions enriched for single nucleotide polymorphisms to allow detection of tumor DNA using the rolling B-allele method. We tested associations with overall survival (OS) and progression-free survival (PFS). RESULTS: Plasma tumor DNA detection was associated with shorter OS [hazard ratio (HR): 2.89, 95% confidence intervals (CI): 1.77-4.73, P ≤ 0.0001] and PFS (HR: 2.05; 95% CI: 1.36-3.11, P < 0.001). Using a multivariable model including plasma tumor DNA, patients who had a TP53, RB1 or PTEN gene alteration pre-treatment and at C2D1 had a significantly shorter OS than patients with no alteration at either time point (TP53: HR 7.13, 95% CI 2.37-21.47, P < 0.001; RB1: HR 6.24, 95% CI 1.97-19.73, P = 0.002; PTEN: HR 11.9, 95% CI 3.6-39.34, P < 0.001). Patients who were positive pre-treatment and converted to undetectable had no evidence of a difference in survival compared with those who were undetectable pre-treatment (P = 0.48, P = 0.43, P = 0.5, respectively). Progression samples harbored AR gain in all patients who had gain pre-treatment (9/49) and de novo AR somatic point mutations were detected in 8/49 patients. CONCLUSIONS: Plasma gene testing after one cycle treatment refines prognostication and could provide an early indication of treatment benefit.

Detecting new microRNAs in human osteoarthritic chondrocytes identifies miR-3085 as a human, chondrocyte-selective, microRNA.

OBJECTIVE: To use deep sequencing to identify novel microRNAs (miRNAs) in human osteoarthritic cartilage which have a functional role in chondrocyte phenotype or function. DESIGN: A small RNA library was prepared from human osteoarthritic primary chondrocytes using in-house adaptors and analysed by Illumina sequencing. Novel candidate miRNAs were validated by northern blot and qRT-PCR. Expression was measured in cartilage models. Targets of novel candidates were identified by microarray and computational analysis, validated using 3'-UTR-luciferase reporter plasmids. Protein levels were assessed by western blot and functional analysis by cell adhesion. RESULTS: We identified 990 known miRNAs and 1621 potential novel miRNAs in human osteoarthritic chondrocytes, 60 of the latter were expressed in all samples assayed. MicroRNA-140-3p was the most highly expressed microRNA in osteoarthritic cartilage. Sixteen novel candidate miRNAs were analysed further, of which six remained after northern blot analysis. Three novel miRNAs were regulated across models of chondrogenesis, chondrocyte differentiation or cartilage injury. One sequence (novel #11), annotated in rodents as microRNA-3085-3p, was preferentially expressed in cartilage, dependent on chondrocyte differentiation and, in man, is located in an intron of the cartilage-expressed gene CRTAC-1. This microRNA was shown to target the ITGA5 gene directly (which encodes integrin alpha5) and inhibited adhesion to fibronectin (dependent on alpha5beta1 integrin). CONCLUSION: Deep sequencing has uncovered many potential microRNA candidates expressed in human cartilage. At least three of these show potential functional interest in cartilage homeostasis and osteoarthritis (OA). Particularly, novel #11 (microRNA-3085-3p) which has been identified for the first time in man.

Hereditary renal amyloidosis associated with a mutant fibrinogen alpha-chain.

Three members of a family who died with renal amyloidosis were found to share a single nucleotide substitution in the fibrinogen alpha-chain gene. The predicted arginine to leucine mutation (Arg554Leu) was proven by amino acid sequence analysis of amyloid fibril protein isolated from postmortem kidney of an affected individual. Direct genomic DNA sequencing and restriction fragment length polymorphism analysis demonstrated that all three affected individuals had the guanine to thymine 4993 transversion. This is the first demonstration of hereditary amyloidosis associated with a variant fibrinogen alpha-chain. Variants of circulating fibrinogen may be the cause of a number of systemic amyloidoses with primarily renal involvement.

Adjustment to cancer and the information needs of people with lung cancer.

OBJECTIVE: Although typically high, the need for information varies between cancer patients. Few studies, however, have examined the factors that predict patient information needs. This study investigated the influence of different styles of adjustment to cancer on information needs. It was proposed that adjustment styles can be defined in terms of goal pursuit and that adjustment influences information needs as these also arise from goal pursuit. METHOD: Seventy-three lung cancer patients were recruited at their first appointment with their radiation oncologist. Participants completed the Patient Information Needs Questionnaire measuring Disease Orientated (DO) information and Action Orientated (AO) information, the Mini-Mental Adjustment to Cancer Scale, and a purpose-built measure of cancer-related personal goals. RESULTS: High levels of the adjustment styles, Fighting Spirit and Anxious Preoccupation, were related to a high need for DO information (p=0.042 and 0.023, respectively). Conversely, high levels of the adjustment style Cognitive Avoidance was related to a low need for DO information (p=0.041). High levels of Anxious Preoccupation were also positively related to a high need for AO information (p=0.018). Support for the proposed theoretical model was also found: information goals predicted information needs and mediated the relationship between Fighting Spirit and DO information need. CONCLUSIONS: These findings suggest that information needs vary as a function of adjustment to cancer. Consequently information provision to cancer patients could be more appropriately tailored by attending to how a patient is adjusting to their diagnosis of cancer.

Guidance of magnetic resonance imaging and placement of skin-marker localization devices.

AIM: A variety of magnetic resonance imaging (MRI)-compatible skin-marker localization devices are available on the market. MRI protocols call for the liberal use of the skin markers over the specific site of symptoms or over any palpable mass. This study investigates the usefulness of patient-assisted placement of 1 000-mg fish oil capsules as skin markers over the area of maximum localized pain, signs, or symptoms and correlates this placement with any potential underlying neuropathology or potential pain generator. METHODS: One-hundred symptomatic patients undergoing MRI were assessed for focal or localized signs or symptoms. Under the direction of a physician and with guidance from the patient, the MRI technician placed a 1 000-mg fish-oil capsule over the area of maximum pain or signs and symptoms. Patients with poorly localized, diffuse symptoms or an area of maximal signs and symptoms outside the field of view of the MRI were not included in this study. All MRI exams were reviewed by clinical physicians and radiologists or neuroimaging physicians. RESULTS: In all 100 cases, the images show clearly visible MRI-compatible skin-surface markers that correlate with potential underlying neuropathology. CONCLUSION: Our results show that 1 000-mg fish-oil capsules can be used as MRI localization devices as a cost-effective alternative to more expensive commercially available devices.

Evaluation of the Impact of Magnetic Resonance Imaging with Susceptibility-weighted Imaging for Screening and Surveillance of Radiation-induced Cavernomas in Long-term Survivors of Malignancy.

AIMS: Radiation-induced cavernomas (RIC) are common late toxicities in long-term survivors of malignancy following cerebral irradiation. However, the natural history of RIC is poorly described. We report the first series of long-term surveillance of RIC using modern magnetic resonance imaging (MRI) including highly sensitive susceptibility-weighted imaging (SWI). The aims of this research were to better characterise the natural history of RIC and investigate the utility of MRI-SWI for screening and surveillance. MATERIALS AND METHODS: Eligibility required long-term survivors of malignancy with previous exposure to cerebral irradiation and RIC identified on MRI-SWI surveillance. The number and size of RIC were reported on Baseline MRI-SWI and last Follow-up MRI-SWI. RESULTS: In total, 113 long-term survivors with RIC underwent MRI-SWI surveillance; 109 (96%) were asymptomatic at the time of RIC diagnosis. The median age at cerebral irradiation was 9.3 years; the median radiotherapy dose was 50.4 Gy. The median time from cerebral irradiation to Baseline MRI-SWI was 17.9 years. On Baseline MRI-SWI, RIC multiplicity was present in 89% of patients; 34% had >10 RIC; 65% had RIC ≥4 mm. The median follow-up from Baseline MRI-SWI was 7.3 years. On Follow-up MRI-SWI, 96% of patients had multiple RIC; 62% had >10 RIC; 72% had RIC ≥4 mm. Of the 109 asymptomatic patients at RIC diagnosis, 96% remained free from RIC-related symptoms at 10 years. Only two required neurosurgical intervention for RIC; there was no RIC-related mortality. CONCLUSIONS: RIC are commonly multiple, asymptomatic and typically increase in size and number over time. Our findings suggest that MRI-SWI for screening of RIC is unlikely to influence longer term intervention in asymptomatic cancer survivors. In the absence of neurological symptoms, assessment or monitoring of RIC are insufficient indications for MRI-SWI surveillance for long-term survivors of malignancy with past exposure to cerebral irradiation.

Long-term Survival with 18-Fluorodeoxyglucose Positron Emission Tomography-directed Therapy in Non-small Cell Lung Cancer with Synchronous Solitary Brain Metastasis.

AIMS: At diagnosis, <1% of patients with non-small cell lung cancer (NSCLC) have synchronous solitary brain metastasis (SSBM). In prior cohorts without 18-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) staging, definitive treatment to intracranial and intrathoracic disease showed a 5-year overall survival (OS) of 11-21%. We investigated the long-term survival outcomes for patients with SSBM NSCLC, diagnosed in the FDG-PET/CT era and treated definitively with local therapies to both intracranial and intrathoracic sites of disease. MATERIALS AND METHODS: This retrospective study assessed patients staged with FDG-PET/CT who received definitive lung and SSBM treatment from February 1999 to December 2017. A lung-molecular graded prognostic assessment (lung-molGPA) score was assigned for each patient using age, performance status score, and, where carried out, molecular status. Overall survival and progression-free survival (PFS) were calculated using Kaplan-Meier methods. Cox proportional hazard models determined OS and PFS prognostic factors. RESULTS: Forty-nine patients newly diagnosed with NSCLC and SSBM had a median age of 63 years (range 34-76). The median follow-up of all patients was 3.9 years. Thirty-three patients (67%) had ≥T2 disease, 23 (47%) had ≥N2. At 2 years, 45% of first failures were intracranial only (95% confidence interval 30-59). At 3 and 5 years, OS was 45% (95% confidence interval 32-63) and 30% (95% confidence interval 18-51), respectively. In ≥N1 disease, 5-year OS was 34% (95% confidence interval 18-63). The 3- and 5-year PFS was 8% (95% confidence interval 3-22) and 0%, respectively. Higher lung-molGPA was associated with longer OS (hazard ratio 0.26, 95% confidence interval 0.11-0.61, P = 0.002). Higher lung-molGPA (hazard ratio 0.33, 95% confidence interval 0.15-0.71, P = 0.005) and lower N-stage (hazard ratio 1.56, 95% confidence interval 1.13-2.15, P = 0.007) were associated with longer PFS. CONCLUSIONS: Definitive treatment of patients with NSCLC and SSBM staged with FDG-PET/CT can result in 5-year survivors, including those with ≥N1 disease.

Differential Response by Melaleuca quinquenervia Trees to Attack by the Rust Fungus Puccinia psidii in Florida.

Melaleuca quinquenervia (melaleuca) is an exotic invasive tree in Florida, Hawaii, and some Caribbean islands (1,2). Puccinia psidii (rust fungus) attacks melaleuca as well as other plants in a few genera of the Myrtaceae and Heteropyxidaceae, both members of the Myrtales (1,2). Disease occurs on succulent stems and foliage of melaleuca, causing twig dieback and defoliation (3). Melaleuca trees growing under similar field conditions exhibit susceptible or resistant reactions toward this fungus. To document this differential susceptibility of melaleuca to P. psidii, we visually evaluated 331 field-grown melaleuca trees from southeast Florida for occurrence of disease attributes: pustules (susceptible), nonpersistent halos (resistant), or asymptomatic (no macroscopic symptoms) conditions on leaves and succulent twigs during February and March when symptoms were at their peak. Percentages of trees manifesting susceptible, resistant, and asymptomatic responses to this fungus were 85.8, 13.0, and 1.2%, respectively. A screenhouse study was conducted to corroborate these observations by raising plants from composite seed sources and maintaining them in seven 3.8-liter plastic pots that were filled with commercial potting media. Nine to eleven plants per pot (with new foliage) were individually tagged, grown to 30 to 45 cm high, and spray inoculated (during February and March) with uredospores (~2 × 106/ml) obtained from melaleuca trees and suspended in water. Inoculated plants were placed on a screenhouse bench under infected trees and subjected to additional inoculum, thereby simulating field conditions. Evaluations made weekly during a 4-week period revealed that susceptible, resistant, and asymptomatic seedlings constituted 63.3, 33.6, and 3.2%, respectively, of the tagged plants. To assess the stability of these fungal and host attributes over time and space, we multiplied two P. psidii susceptible and two resistant plants from cuttings. We spray inoculated 6 to 13 rooted cuttings from each plant types with uredospores (0.8 to 2 × 106/ml) obtained from diseased melaleuca trees and suspended in water. These plants were incubated in a dew chamber for 72 to 96 h under 100% relative humidity at 19 to 23°C maintained with a 12-h fluorescent light cycle. After incubation, plants were placed randomly on a bench in a screenhouse (21 to 23°C) and evaluated weekly for symptom development during a 4-week experimental period. Noninoculated controls were maintained as well. The experiment was repeated twice. Foliage of the resistant plants developed a few incipient halos whereas 100% of the susceptible plants developed erupted uredinia and were defoliated in both replications. No detectable change in P. psidii virulence and melaleuca susceptibility patterns was observed. Despite wide host range within Myrtales, resistance to P. psidii exists within M. quinquenervia. Other P. psidii susceptible host systems of economic and environmental importance may have host/pathogen relationships similar to that of melaleuca and the selection of resistant individuals from their affected populations may be possible. Additional studies will be needed to ascertain the attributes of virulence or resistance in this rust fungus-melaleuca association. References: (1) M. Glen et al. Australas. Plant Pathol. 36:1, 2007. (2) P. D. Pratt et al. J. Aquat. Plant Manag. 45:8, 2007. (3) M. B. Rayachhetry et al. Biol. Control 22:38, 2001.

Binding of integrin alpha6beta4 to plectin prevents plectin association with F-actin but does not interfere with intermediate filament binding.

Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the alpha6beta4 integrin and have shown that the cytoplasmic domain of the beta4 subunit associates with an NH(2)-terminal fragment of plectin that contains the actin-binding domain (ABD). When expressed in immortalized plectin-deficient keratinocytes from human patients with epidermol- ysis bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is colocalized with alpha6beta4 in basal hemidesmosome-like clusters or associated with F-actin in stress fibers or focal contacts. We used a yeast two-hybrid binding assay in combination with an in vitro dot blot overlay assay to demonstrate that beta4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the beta4 cytoplasmic domain. Mapping of the beta4 and actin-binding sites on plectin showed that the binding sites overlap and are both located in the plectin ABD. Using an in vitro competition assay, we could show that beta4 can compete out the plectin ABD fragment from its association with F-actin. The ability of beta4 to prevent binding of F-actin to plectin explains why F-actin has never been found in association with hemidesmosomes, and provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament-anchoring hemidesmosomes when beta4 is expressed. Finally, by mapping of the COOH-terminally located binding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EBS keratinocytes, we confirm that plectin interacts with different cytoskeletal networks.

Unravelling the mechanisms that determine the uptake and metabolism of magnetic single and multicore nanoparticles in a Xenopus laevis model.

Multicore superparamagnetic nanoparticles have been proposed as ideal tools for some biomedical applications because of their high magnetic moment per particle, high specific surface area and long term colloidal stability. Through controlled aggregation and packing of magnetic cores it is possible to obtain not only single-core but also multicore and hollow spheres with internal voids. In this work, we compare toxicological properties of single and multicore nanoparticles. Both types of particles showed moderate in vitro toxicity (MTT assay) tested in Hep G2 (human hepatocellular carcinoma) and Caco-2 (human colorectal adenocarcinoma) cells. The influence of surface chemistry in their biological behavior was also studied after functionalization with O,O'-bis(2-aminoethyl) PEG (2000 Da). For the first time, these nanoparticles were evaluated in a Xenopus laevis model studying their whole organism toxicity and their impact upon iron metabolism. The degree of activation of the metabolic pathway depends on the size and surface charge of the nanoparticles which determine their uptake. The results also highlight the potential of Xenopus laevis model bridging the gap between in vitro cell-based assays and rodent models for toxicity assessment to develop effective nanoparticles for biomedical applications.

Single Fraction Stereotactic Ablative Body Radiotherapy for Oligometastasis: Outcomes from 132 Consecutive Patients.

AIMS: Stereotactic ablative body radiotherapy (SABR) is currently used to treat oligometastases, but the optimum dose/fractionation schedule is unknown. In this study, we evaluated outcomes after single fraction SABR in patients with oligometastatic disease. MATERIALS AND METHODS: Single institutional retrospective review of patients treated with single fraction SABR for one to three oligometastases between 2010 and 2015. The primary outcome was freedom from widespread disease defined as distant recurrence not amenable to surgery or SABR; or recurrence with four or more metastases. RESULTS: In total, 186 treatments were delivered in 132 patients. The two most common target sites were lung (51%) and bone (40%). The most frequent single fraction prescription dose was 26 Gy (47%). The most common primary malignancy was genitourinary (n = 46 patients). Freedom from widespread disease was 75% at 1 year (95% confidence interval 67-83%) and 52% at 2 years (95% confidence interval 42-63%). Freedom from local progression at 1 year was 90% (95% confidence interval 85-95%) and at 2 years was 84% (95% confidence interval 77-91%). A compression fracture of the lumbar vertebra was the only grade 3+ treatment-related toxicity. CONCLUSIONS: Single fraction SABR is associated with a high rate of freedom from widespread disease, favourable local control and low toxicity comparable with historic multi-fraction SABR reports.

Inducible gene expression in transgenic Xenopus embryos.

The amphibian Xenopus laevis has been successfully used for many years as a model system for studying vertebrate development. Because of technical limitations, however, molecular investigations have mainly concentrated on early stages. We have developed a straightforward method for stage-specific induction of gene expression in transgenic Xenopus embryos [1] [2]. This method is based on the Xenopus heat shock protein 70 (Xhsp70 [3]) promoter driving the expression of desired gene products. We found that ubiquitous expression of the transgene is induced upon relatively mild heat treatment. Green fluorescent protein (GFP) was used as a marker to monitor successful induction of gene expression in transgenic embryos. We used this method to study the stage specificity of Wnt signalling function. Transient ectopic Wnt-8 expression during early neurulation was sufficient to repress anterior head development and this capacity was restricted to early stages of neurulation. By transient over-expression at different stages of development, we show that frizzled-7 disrupted morphogenesis sequentially from anterior to posterior along the dorsal axis as development proceeds. These results demonstrate that this method for inducible gene expression in transgenic Xenopus embryos will be a very powerful tool for temporal analysis of gene function and for studying molecular mechanisms of vertebrate organogenesis.

Hydrilla stems and tubers as hosts for three Bagous species: two introduced biological control agents (Bagous hydrillae and B. affinis) and one native species (B. restrictus).

Field observations suggested that the introduced Hydrilla verticillata (L.f.) Royle biological control agent, a stem weevil, Bagous hydrillae O'Brien, would feed on hydrilla tubers and stems, and a native species, Bagous restrictus LeConte, would feed on hydrilla stems. In choice tests, B. hydrillae readily oviposited in hydrilla tubers. Larval development of B. hydrillae in hydrilla tubers was similar to that in stems; greater adult biomass was attained and less time was needed to complete development when the larvae were fed tubers. Larvae of the hydrilla tuber weevil, B. affinis Hustache, did not complete development in hydrilla stems. Larvae of B. affinis completed development more rapidly when fed new compared with old hydrilla tubers. The native B. restrictus successfully completed development in hydrilla stems, although the larvae required slightly more time compared with the biocontrol agent, B. hydrillae. These findings indicated that feeding on tubers by B. hydrillae may benefit the species particularly when hydrilla stems are seasonably absent or unsuitable especially in more northern climates. In terms of hydrilla control, damage to tubers by this species constitutes a reduction in future infestations of hydrilla propagated by tubers. Finally, hydrilla is suitable to the native weevil, B. restrictus, because larvae completed development in hydrilla stems.

Impact of Field Densities of the Naturalized Defoliator Caloptilia triadicae (Lepidoptera: Gracillariidae) on the Invasive Weed Chinese Tallowtree.

Chinese tallowtree, Triadica sebifera (L.) Small (Euphoriaceae), is one of the most aggressive weeds of coastal wetlands and forests of the southeastern United States. The lack of specialist herbivores in the invaded range may be one of the factors that contribute to the invasive nature of this weed. Chinese tallowtree has been the target of a classical biological control project since 2006. Several herbivore species are being tested for biological control of Chinese tallowtree. Concurrently, an adventive herbivore, Caloptilia triadicae Davis (Lepidoptera: Gracillariidae), was found feeding on Chinese tallowtree in the southeastern United States in 2004 and now occurs throughout the invaded range. Field populations of C. triadicae from two sites caused extensive mining damage to the Chinese tallowtree leaves. The greatest damage occurred after 30 d of exposure to C. triadicae in the herbivory treatment and amounted to about 25-30 leaf mines (early instars) and leaf rolls (late instars) per plant. Insecticide-treated plants had atmost 5-10 leaf mines and rolls per plant. Despite this difference, plant growth in height, number of new branches, and leaves did not differ significantly from plants protected from herbivory. Analysis of harvested plant results suggested that, in general, herbivory had little impact on biomass. However, there was a slight reduction in trunk weights in the unrestriced herbivory treatment compared with the insecticide-treated plants. Although this study exposed experimental plants to only 60 d of herbivory, these results suggested that C. triadicae alone will not exert sufficient control of invasive populations of Chinese tallowtree. Furthermore, they indicated that continued development of biological control agents that target Chinese tallowtree are needed.

Management of paediatric thyroid carcinoma: recent experience with recombinant human thyroid stimulating hormone in preparation for radioiodine therapy.

BACKGROUND: Thyroid carcinoma in children is rare and raises unique management issues. Although metastatic disease is more common in this age group, prognosis remains good with appropriate treatment. The aim of the study was to report recent experience in the management of differentiated thyroid carcinoma in children, especially in the use of radioiodine after recombinant human thyroid stimulating hormone (rhTSH) stimulation. METHODS: Eight patients, aged 5-17 years (five were boys) presented following total thyroidectomy for thyroid carcinoma between May 2003 and June 2005. Seven had papillary carcinoma and one had follicular carcinoma. Five had known lymph node metastases and one had pulmonary metastases at presentation. Four patients had previously received therapeutic irradiation for malignancy. All eight underwent diagnostic iodine scans, seven with rhTSH stimulation. Seven went on to receive radioiodine treatment as hospital inpatients, comanaged by the paediatric and nuclear medicine units. The dosage of 131I ranged from 1.5 to 3.7 x 10(9) Bq. All except one were prepared by rhTSH stimulation. RESULTS: Seven of eight patients had significant uptake in the neck on diagnostic scan and two had pulmonary abnormalities. Six of seven evaluable patients achieved complete thyroid ablation. Both patients with pulmonary abnormalities had scan resolution, although one of them only after a second radioiodine treatment. All patients had thyroxine replacement in doses to suppress TSH and all remain alive and well at time of carrying out this study. CONCLUSION: Optimal management of paediatric thyroid carcinoma necessitates a multidisciplinary approach. Radioiodine therapy under rhTSH is an effective and safe adjuvant treatment in this special subgroup.

DNA damage in cultured L1210 cells by 2-haloethyl esters of (methylsulfonyl)methanesulfonic acid.

The effects of the 2-chloroethyl, 2-bromoethyl, and 2-fluoroethyl esters of (methylsulfonyl)methanesulfonic acid upon the DNA of cultured L1210 cells have been measured and compared with each other and with the effects of chlorozotocin. Results obtained by the alkaline elution method indicated that, at equimolar and equitoxic concentrations, the esters caused more strand scission than chlorozotocin, but at compound concentrations that caused a 50% reduction in colony formation by cells following an exposure period of 2 h, they caused no detectable cross-linking, whereas chlorozotocin did cause cross-linking. Two in vitro experimental methods that are based upon the complexing of ethidium to calf thymus DNA also yielded data showing that, at equimolar concentrations, chlorozotocin caused cross-linking of calf thymus DNA, but the 2-chloroethyl ester did not. These results indicate that these esters might not kill cells by producing DNA-DNA cross-links. The three esters caused qualitatively similar effects, but the fluoro esters caused less strand scission than the chloro and bromo esters, which caused about the same extent of strand scission.

Effects of N,N'-bis(2-chloroethyl)-N-nitrosourea and 2-[3-(2-chloroethyl)-3-nitrosoureido]-2-deoxy-D-glucopyranose upon the proliferation, DNA synthesis, and viability of cultured sensitive and resistant L1210 cells.

The effects of N,N'-bis(2-chloroethyl)-N-nitrosourea and chlorozotocin upon the proliferation, DNA synthesis, and viability of cultured cells of a sensitive line of L1210 leukemia, a line partially resistant to N,N'-bis(2-chloroethyl)-N-nitrosourea, and a line resistant to cyclophosphamide were determined. The results indicate that neither the effect upon proliferation nor the effect upon DNA synthesis is a good predictor of the extent of cell kill. The similarity of the effects of N,N'-bis(2-chloroethyl)-N-nitrosourea upon these two parameters for the three cell lines indicates that the sensitive and resistant cells are affected to approximately the same extent, but more of the resistant cells survive. Additional studies are required to seek the reasons for this differential survival.

Carbamoylation of amino acid, peptides, and proteins by nitrosoureas.

Incubation at approximately physiological conditions of amino acids, peptides, and proteins with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea or cyclohexyl isocyanate resulted in carbamoylation of the alpha-amino groups of amino acids, the terminal amino groups of peptides and proteins and the epsilon-amino groups of lysine moieties. Carbamoylation of the alpha-amino groups and the terminal amino groups occurred as readily as, or more readily than, the carbamoylation of the epsilon-amino groups. Carbamoylation of the amino groups of amino acids or peptides by 1,3-bis(2-chloroethyl)-1-nitrosourea or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea altered the electrophoretic mobility of those compounds. Cyclization of (2-cloroethylcarbamoyl)-amino groups to form (2-oxazolin-2-yl)amino groups occurred at room temperature, and the resulting oxazolinyl compounds migrated electrophoretically similarly to the parent compounds. Since such cyclization did not occur with cyclohexylcarbamoylamino groups, treatment of amino acids, peptides, or proteins with 1,3-bis(2-chloroethyl)-1-nitrosourea might result in less permanent alteration of the respective charges on the resulting products than would treatment with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea or other nitrosoureas lacking a 2-chloroethyl group on N-3. The relevance of these differences in charge to differences in physiological effects is not presently known. Although the present study does not establish a definite relationship between carbamoylation of any specific protein and the physiological effects of nitrosourea, it does reinforce and expand the existing evidence that carbamoylation of proteins is a proteins is a process that must be considered in efforts to explain the physiological effects of these agents, and it points to terminal amino groups of proteins as possible primary sites of carbamoylation.

Inhibition of rat liver DNA polymerase by nitrosoureas and isocyanates.

The effects of 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexl-1-nitrosourea, and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea on two nonmitochondrial DNA polymerases (I and II) purified from rat liver and hepatoma were examined. The activity of DNA polymerase I was not altered by treatment with any of the nitrosoureas or the corresponding isocyanates, 2-chloroethyl isocyanate and cyclohexyl isocyanate. Incubation of DNA polymerase II with the nitrosoureas (1 mM) inhibited its enzymatic activity 30 to 45%. DNA polymerase II was inhibited 75 and 90% by 1.mM 2-chloroethyl isocyanate and cyclohexyl isocyanate, respectively. The nitrosoureas appear to exert their inhabitory action on the enzyme (DNA polymerase II) rather than on the DNA template. Pretreatment of the enzyme increased the degree of inhibition by 1 mM nitrosourea (50 to 60% inhibition) or 2-chloroethul isocyanate (greater than 90% inhibition), whereas pretreatment of the DNA template did not enhance the inhibitory effect. The three nitrosoureas are equally effective as inhibitors of DNA polymerase II. 2-Chloroethyl isocyanate and cyclohexyl isocyanate are better inhibitors than are the nitrosoureas. Since further decomposition products of the isocyanates, 2-chloroethylamine and cyclohexylamine, do not inhibit DNA polymerase II, we conclude that the isocyanates, which are decomposition products of the nitrosoureas, are the active inhibitors of the enzyme.

Comparison of 1,2-dihydropyrido[3,4-b]pyrazines (1-deaza-7,8-dihydropteridines) with several other inhibitors of mitosis.

Several properties of four 1-deaza-7,8-dihydropteridines were compared with those of each other and with those of colchicine, nocodazole, podophyllotoxin, and vincristine. Compound NSC 370147 was more active than the other compounds of this type with respect to inhibition of proliferation of cultured L1210 cells and to increase of the mitotic index. On an equimolar basis it was more active than two of the 1-deaza-7,8-dihydropteridines, colchicine, and nocodazole and was comparable to podophyllotoxin and vincristine in inhibiting the polymerization of partially purified pig brain tubulin. All four of the 1-deaza-7,8-dihydropteridines caused decreases in the extent of binding of [3H]colchicine to partially purified tubulin and enhanced the binding of [3H]vincristine to the tubulin. Emphasis in further testing was placed upon NSC 370147, because it is easier to synthesize and is more stable than some of the other compounds of this type and because its greater solubility in water facilitates its formulation for therapeutic administration. Compound NSC 370147 inhibited competitively the binding of [3H]colchicine to purified tubulin and enhanced slightly the binding of [3H]vincristine to tubulin. It was also synergistic with vincristine in killing cultured L1210 cells and in increasing the life-spans of mice bearing P388 leukemia. It is suggested that it would be worthwhile to evaluate combinations of NSC 370147 and vincristine in tests with other experimental neoplasms.