Further studies with a cell immortalization assay to investigate the mutation signature of aristolochic acid in human p53 sequences

Feldmeyer, Nadja; Schmeiser, Heinz H.; Muehlbauer, Karl-Rudolf; Belharazem, Djeda; Knyazev, Yuri P.; Nedelko, Tatiana; Hollstein, Monica

doi:10.1016/j.mrgentox.2006.02.017

Cited by 52 publications

(73 citation statements)

References 21 publications

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“…The p53 mutational spectra of these cancers are dominated by A:T 3 T:A transversions, resembling the mutational ''signature'' observed in cultured cells and rodents treated with AA (23)(24)(25)(26)(27)(28). These findings provide support for our guiding hypothesis that dietary exposure to AA is a risk factor for EN and that AA-derived DNA adducts initiate the transitional cell cancers associated with this disease.…”

supporting

confidence: 73%

“…This result is consistent with the mutational spectra induced by AA-I (or by a mixture of AA-I and AA-II) in (i) the H-ras gene of rats (23,24), (ii) transgenic rodent models (25,26), (iii) the p53 gene in a urothelial cancer from a patient with AAN (34), (iv) site-specific mutagenesis studies in which a single dA-AL adduct is transfected into NER-deficient human cells (I.-Y. Yang and M.M., unpublished data), and (v) 9 of 11 immortalized mouse cell lines (Hupki cells) carrying the human p53 gene (27,28). Indeed, the predominance of A:T 3 T:A transversions in the p53 mutational spectra may properly be regarded as a mutational signature (35) for human exposure to AA.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, building on epidemiologic, environmental, and agricultural evidence (6,(16)(17)(18)(19), we examined renal tissues of EN patients for these biomarkers of exposure to AA. Additionally, AL-DNA adducts are known to be mutagenic (23)(24)(25)(26)(27)(28), leading us to determine the p53 mutational spectrum of transitional cell cancers in patients with EN.…”

mentioning

confidence: 99%

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Aristolochic acid and the etiology of endemic (Balkan) nephropathy

Grollman

Shibutani

Moriya

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

522

590

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supporting

confidence: 73%

Section: Discussionmentioning

confidence: 99%

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Aristolochic acid and the etiology of endemic (Balkan) nephropathy

Grollman

Shibutani

Moriya

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

522

590

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“…The same type of mutation was found in TP53 in urothelial tumour cells of an AAN patient (Lord et al, 2004), suggesting that TP53 is mutated in AAN-associated tumours (Arlt et al, 2001b;Arlt et al, 2007b). Furthermore, AT→TA transversions were found in TP53 of immortalised cells derived from murine primary human TP53 knock-in (Hupki) embryonic fibroblasts exposed to AAI (Liu et al, 2004;Feldmeyer et al, 2006;vom Brocke et al, 2006). Interestingly, in one cell line the mutation was in codon 139, identical to one found in the urothelial tumour cells of the AAN patient (Lord et al, 2004;Feldmeyer et al, 2006).…”

Section: Discussionmentioning

confidence: 83%

Gene expression profiles modulated by the human carcinogen aristolochic acid I in human cancer cells and their dependence on TP53

Simões

Hockley

Schwerdtle

et al. 2008

Toxicology and Applied Pharmacology

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Aristolochic acid (AA) is the causative agent of urothelial tumours associated with aristolochic acid nephropathy. These tumours contain TP53 mutations and over-express TP53. We compared transcriptional and translational responses of two isogenic HCT116 cell lines, one expressing TP53 (p53-WT) and the other with this gene knocked out (p53-null), to treatment with aristolochic acid I (AAI) (50−100 µM) for 6−48 h. Modulation of 118 genes was observed in p53-WT cells and 123 genes in p53-null cells. Some genes, including INSIG1, EGR1, CAV1, LCN2 and CCNG1, were differentially expressed in the two cell lines. CDKN1A was selectively up-regulated in p53-WT cells, leading to accumulation of TP53 and CDKN1A. Apoptotic signalling, measured by caspase-3 and -7 activity, was TP53-dependent. Both cell types accumulated in S phase, suggesting that AAI-DNA adducts interfere with DNA replication, independently of TP53 status. The oncogene MYC, frequently over-expressed in urothelial tumours, was up-regulated by AAI, whereas FOS was downregulated. Observed modulation of genes involved in endocytosis, e.g. RAB5A, may be relevant to the known inhibition of receptor-mediated endocytosis, an early sign of AA-mediated proximal tubule injury. AAI-DNA adduct formation was significantly greater in p53-WT cells than in p53-null cells. Collectively, phenotypic anchoring of the AAI-induced expression profiles to DNA adduct formation, cell-cycle parameters, TP53 expression and apoptosis identified several genes linked to these biological outcomes, some of which are TP53-dependent. These results strengthen the importance of TP53 in AA-induced cancer, and indicate that other alterations, e.g. to MYC oncogenic pathways, may also contribute.

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“…DNA from the immortalised HUFs is then sequenced to identify TP53 mutations. Previous studies have detected TP53 mutations in up to 30% of mutagen‐treated cultures or 5 to 20% of spontaneously immortalised cultures 10, 11, 12, 13. The remaining cultures are TP53 ‐WT and likely harbour alterations in other genes associated with senescence‐bypass 14, 15…”

mentioning

confidence: 99%

Nutlin‐3a selects for cells harbouring TP53 mutations

Kucab

Hollstein

Arlt

et al. 2016

Intl Journal of Cancer

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TP53 mutations occur in half of all human tumours. Mutagen‐induced or spontaneous TP53 mutagenesis can be studied in vitro using the human TP53 knock‐in (Hupki) mouse embryo fibroblast (HUF) immortalisation assay (HIMA). TP53 mutations arise in up to 30% of mutagen‐treated, immortalised HUFs; however, mutants are not identified until TP53 sequence analysis following immortalisation (2–5 months) and much effort is expended maintaining TP53‐WT cultures. In order to improve the selectivity of the HIMA for HUFs harbouring TP53 mutations, we explored the use of Nutlin‐3a, an MDM2 inhibitor that leads to stabilisation and activation of wild‐type (WT) p53. First, we treated previously established immortal HUF lines carrying WT or mutated TP53 with Nutlin‐3a to examine the effect on cell growth and p53 activation. Nutlin‐3a induced the p53 pathway in TP53‐WT HUFs and inhibited cell growth, whereas most TP53‐mutated HUFs were resistant to Nutlin‐3a. We then assessed whether Nutlin‐3a treatment could discriminate between TP53‐WT and TP53‐mutated cells during the HIMA (n = 72 cultures). As immortal clones emerged from senescent cultures, each was treated with 10 µM Nutlin‐3a for 5 days and observed for sensitivity or resistance. TP53 was subsequently sequenced from all immortalised clones. We found that all Nutlin‐3a‐resistant clones harboured TP53 mutations, which were diverse in position and functional impact, while all but one of the Nutlin‐3a‐sensitive clones were TP53‐WT. These data suggest that including a Nutlin‐3a counter‐screen significantly improves the specificity and efficiency of the HIMA, whereby TP53‐mutated clones are selected prior to sequencing and TP53‐WT clones can be discarded.

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Further studies with a cell immortalization assay to investigate the mutation signature of aristolochic acid in human p53 sequences

Cited by 52 publications

References 21 publications

Aristolochic acid and the etiology of endemic (Balkan) nephropathy

Aristolochic acid and the etiology of endemic (Balkan) nephropathy

Gene expression profiles modulated by the human carcinogen aristolochic acid I in human cancer cells and their dependence on TP53

Nutlin‐3a selects for cells harbouring TP53 mutations

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