Adaptation of cancer cells from different entities to the MDM2 inhibitor nutlin-3 results in the emergence of p53-mutated multi-drug-resistant cancer cells

Six p53 wild-type cancer cell lines from infrequently p53-mutated entities (neuroblastoma, rhabdomyosarcoma, and melanoma) were continuously exposed to increasing concentrations of the murine double minute 2 inhibitor nutlin-3, resulting in the emergence of nutlin-3-resistant, p53-mutated sublines displaying a multi-drug resistance phenotype. Only 2 out of 28 sublines adapted to various cytotoxic drugs harboured p53 mutations. Nutlin-3-adapted UKF-NB-3 cells (UKF-NB-3^rNutlin^10 μM, harbouring a G245C mutation) were also radiation resistant. Analysis of UKF-NB-3 and UKF-NB-3^rNutlin^10 μM cells by RNA interference experiments and lentiviral transduction of wild-type p53 into p53-mutated UKF-NB-3^rNutlin^10 μM cells revealed that the loss of p53 function contributes to the multi-drug resistance of UKF-NB-3^rNutlin^10 μM cells. Bioinformatics PANTHER pathway analysis based on microarray measurements of mRNA abundance indicated a substantial overlap in the signalling pathways differentially regulated between UKF-NB-3^rNutlin^10 μM and UKF-NB-3 and between UKF-NB-3 and its cisplatin-, doxorubicin-, or vincristine-resistant sublines. Repeated nutlin-3 adaptation of neuroblastoma cells resulted in sublines harbouring various p53 mutations with high frequency. A p53 wild-type single cell-derived UKF-NB-3 clone was adapted to nutlin-3 in independent experiments. Eight out of ten resulting sublines were p53-mutated harbouring six different p53 mutations. This indicates that nutlin-3 induces de novo p53 mutations not initially present in the original cell population. Therefore, nutlin-3-treated cancer patients should be carefully monitored for the emergence of p53-mutated, multi-drug-resistant cells.     

Mutagenicity and clastogenicity of proflavin in L5178Y/TK +/- -3.7.2.C cells

We evaluated the ability of proflavin to induce specific-locus mutations at the heterozygous thymidine kinase (tk) locus of L5178Y/TK +/- -3.7.2C mouse lymphoma cells, which appears to permit the recovery of mutants due to single-gene and chromosomal mutations. Proflavin was highly mutagenic at the tk locus, producing 724-965 TK mutants/10(6) survivors (background = 56-85/10(6); survival = 29-32%). Most of the mutants were small colonies, which suggested that proflavin may induce chromosomal mutations. The potent clastogenicity of proflavin was confirmed by cytogenetic analysis for chromosomal aberrations. At the highest dose analyzed (1.5 micrograms/ml), proflavin produced 82 aberrations/100 metaphaes (background = 2/100). The large-colony TK mutant frequency produced by proflavin (48-109/10(6) survivors; background = 23/10(6); survival = 57-61%) was similar to published HPRT mutant frequencies produces by proflavin in L5178Y and CHO cells (50-100/10(6) survivors; background = 2-50/10(6); survival = 50-62%). These results lead to the conclusion that proflavin is a potent clastogen and induces a high frequency of small-colony TK mutants; however, it induces a low frequency of HPRT mutants and a low frequency of large-colony TK mutants.     

Mutagenicity and clastogenicity of teniposide (VM-26) in L5178Y/TK +/- -3.7.2C mouse lymphoma cells

The antitumor drug teniposide (VM-26) is a potent inducer of DNA breaks (Long et al., Cancer Res., (1985) 45, 3106), but it is only weakly mutagenic at the hprt locus in CHO cells (Singh and Gupta, Cancer Res., (1983) 43, 577). In the present study, the mutagenic and clastogenic activities of teniposide were evaluated in L5178Y/TK +/- -3.7.2C mouse lymphoma cells. Although teniposide is a weak mutagen at the hprt locus, it is a potent mutagen at the tk locus, with as little as 0.5 ng/ml producing 220 TK mutants/10(6) survivors at 96% survival (background = 100/10(6) survivors). This same dose of teniposide induced 38 aberrations per 100 metaphases (background = 7/100 cells). At 7 ng/ml, teniposide induced approximately 2700 TK mutants/10(6) survivors at approximately 10% survival. At the highest dose sampled for aberration analysis (5 ng/ml), teniposide induced 44 aberrations/100 cells. Most of the aberrations were chromosomal rather than chromatid events. As expected for a compound acting primarily by a clastogenic mechanism, most of the TK mutants were small colonies. Thus, teniposide is a potent clastogen, and it is a potent mutagen at the tk locus but not at the hprt locus. These results support the hypothesis that the location of the target gene affects the ability of the assay to detect both intragenic events and events causing functional multilocus effects. Thus, a heterozygous locus (like tk) but not a functionally hemizygous locus (like hprt) may permit the detection of mutagens that act primarily by a clastogenic mechanism. Because teniposide induces topoisomerase II-associated DNA breaks, and because there is evidence that teniposide may not interact directly with DNA, we discuss the possibility that the potent clastogenic/mutagenic activity of teniposide may be mediated by topoisomerase II.     

Introduction to the Special Issue,Commitment to Alterity and Its Disavowal: The Politics of Display of Belonging

ABSTRACT

As multiple ethnic/race affiliation is highlighted more and more, we lack analytical frameworks to examine diverse ways individuals navigate through them as they balance aspirations, fears, desires, pride, responsibility, and pragmatic necessities. Existing studies of identification practices offer little examination of practices of those who disavow identifying with certain ethnic/race categories except for the ones focused on a narrow field of social relations, such as the academic achievement and career success in ‘acting White.’ This introductory piece introduces the main theoretical ideas in the special issue, commitment to alterity and its disavowal, which expands the scope of analysis to a wider range of identification practices and fields of social relations. This piece also briefly describes each contributing article, which further develops these notions to analyse various contours and degrees of belonging and links to wider cultural politics and power relations.     

‘I Have Māori in Me’: Shades of Commitment and Negotiation of Subjectivity in an Aotearoa/New Zealand School

ABSTRACT

Based on ethnographic fieldwork (1997–1998) at a secondary school with a Māori?English bilingual unit in Aotearoa/New Zealand, this article examines two different ways students with Māori ancestry identified themselves contextually: those in the bilingual unit identified themselves mostly as being Māori, while those in mainstream classes identified themselves mostly as Pākehā (white New Zealander) but occasionally as being Pākehā but having Māori in them. Existing analytical frameworks, such as symbolic ethnicity (Gans 1999) or citizenship (Ong 2003), fail to capture the contextual and dialogic display of these different shades of identification practices. Applying the notion of commitment and its disavowal proposed by Doerr for this special issue (2013), this article analyses these two identification practices as a proactive commitment and a hedging commitment linked to institutional belonging to the bilingual unit and mainstream classes, respectively, and to the wider cultural politics of the official yet tokenistic biculturalism of Aotearoa/New Zealand.     

Unprotected synthesis

Chemists at The Scripps Research Institute demonstrate that it is possible to synthesize complex natural products without the use of protecting groups.     

Positive effects of helpers on reproductive success in the brown treecreeper and the general importance of future benefits

1. Numerous studies of cooperatively breeding species have tested for effects of helpers on reproductive success to evaluate hypotheses for the evolution of cooperation, but relatively few have used experimental or statistical approaches that control for the confounding effects of breeder and territory quality. 2. In the brown treecreeper Climacteris picumnus, most helpers are male offspring of the breeding pair that have delayed dispersal. We analysed 5 years of data (97 territory-years) using hierarchical linear modelling to test for effects of helpers on reproductive success while controlling for confounding factors. 3. The number of helpers was related positively to reproductive success even after controlling for differences between territories and breeders. A threshold effect was observed, with success increasing most with the presence of a second helper (i.e. at group size of four). 4. Feeding at the nest was one mechanism responsible for this effect, as larger groups had higher total feeding rates at all nesting stages. Higher total feeding rates, as well as higher feeding rates by helpers, were correlated in turn with greater reproductive success. 5. An analysis of the effects of helper feeding rate on reproductive success in groups with just one helper produced only weak support for a positive effect of helpers. Controlled comparisons of this kind utilize only a small fraction of the total data available and thus have limited statistical power compared to hierarchical or mixed-modelling. 6. A number of hypotheses to explain the evolution and maintenance of helping behaviour are consistent with our results for brown treecreepers including kin selection and hypotheses based on future direct benefits. 7. A previous synthesis of studies of helper effects that controlled for confounding factors suggested a pattern in which male helpers rarely have positive effects on reproductive success. However, revising that synthesis to include recent hierarchical or mixed-modelling studies suggests that helpers of both sexes usually have positive effects, and that the relative importance of future direct benefits may have been underestimated.     

Mutagenicity and clastogenicity of adriamycin in L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells

Adriamycin was found to be both mutagenic and clastogenic to L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells. A dose of only 5 ng/ml (survival = 62% or 67%) gave an induced TK mutant frequency of 307 or 296 per 10(6) survivors in two separate experiments. This dose was also clastogenic, inducing 20 chromosome aberrations/100 cells analyzed. The majority of the mutants were small-colony mutants, indicating that adriamycin likely acts primarily by a clastogenic mechanism.