Ellen van Drunen

Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells

The use of Cre/loxP recombination in mammalian cells has expanded rapidly. We describe here that Cre expression in cultured mammalian cells may result in a markedly reduced proliferation and that this effect is dependent on the endonuclease activity of Cre. Chromosome analysis after Cre expression revealed numerous chromosomal aberrations and an increased number of sister chromatid exchanges. Titration experiments in mouse embryo fibroblasts with a ligand-regulatable Cre-ERT show that toxicity is dependent on the level of Cre activity. Prolonged, low levels of Cre activity permit recombination without concomitant toxicity. This urges for a careful titration of Cre activity in conditional gene modification in mammalian cells.

The Structure-Specific Endonuclease Ercc1-Xpf Is Required To Resolve DNA Interstrand Cross-Link-Induced Double-Strand Breaks

ABSTRACT Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (γ-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced γ-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, γ-H2AX foci were also induced in Ercc1−/− cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1 −/− cells, MMC-induced γ-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1 −/− and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.

The structure-specific endonuclease Mus81 contributes to replication restart by generating double-strand DNA breaks

Faithful duplication of the genome requires structure-specific endonucleases such as the RuvABC complex in Escherichia coli. These enzymes help to resolve problems at replication forks that have been disrupted by DNA damage in the template. Much less is known about the identities of these enzymes in mammalian cells. Mus81 is the catalytic component of a eukaryotic structure-specific endonuclease that preferentially cleaves branched DNA substrates reminiscent of replication and recombination intermediates. Here we explore the mechanisms by which Mus81 maintains chromosomal stability. We found that Mus81 is involved in the formation of double-strand DNA breaks in response to the inhibition of replication. Moreover, in the absence of chromosome processing by Mus81, recovery of stalled DNA replication forks is attenuated and chromosomal aberrations arise. We suggest that Mus81 suppresses chromosomal instability by converting potentially detrimental replication-associated DNA structures into intermediates that are more amenable to DNA repair.

Selective Inhibition of BRCA2-Deficient Mammary Tumor Cell Growth by AZD2281 and Cisplatin

Purpose: To assess efficacy of the novel, selective poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor AZD2281 against newly established BRCA2-deficient mouse mammary tumor cell lines and to determine potential synergy between AZD2281 and cisplatin. Experimental Design: We established and thoroughly characterized a panel of clonal cell lines from independent BRCA2-deficient mouse mammary tumors and BRCA2-proficient control tumors. Subsequently, we assessed sensitivity of these lines to conventional cytotoxic drugs and the novel PARP inhibitor AZD2281. Finally, in vitro combination studies were done to investigate interaction between AZD2281 and cisplatin. Results: Genetic, transcriptional, and functional analyses confirmed the successful isolation of BRCA2-deficient and BRCA2-proficient mouse mammary tumor cell lines. Treatment of these cell lines with 11 different anticancer drugs or with γ-irradiation showed that AZD2281, a novel and specific PARP inhibitor, caused the strongest differential growth inhibition of BRCA2-deficient versus BRCA2-proficient mammary tumor cells. Finally, drug combination studies showed synergistic cytotoxicity of AZD2281 and cisplatin against BRCA2-deficient cells but not against BRCA2-proficient control cells. Conclusion: We have successfully established the first set of BRCA2-deficient mammary tumor cell lines, which form an important addition to the existing preclinical models for BRCA-mutated breast cancer. The exquisite sensitivity of these cells to the PARP inhibitor AZD2281, alone or in combination with cisplatin, provides strong support for AZD2281 as a novel targeted therapeutic against BRCA-deficient cancers.

High EVI1 levels predict adverse outcome in acute myeloid leukemia: prevalence of EVI1 overexpression and chromosome 3q26 abnormalities underestimated

Inappropriate expression of EVI1 (ecotropic virus integration-1), in particular splice form EVI1-1D, through chromosome 3q26 lesions or other mechanisms has been implicated in the development of high-risk acute myeloid leukemia (AML). To validate the clinical relevance of EVI1-1D, as well as of the other EVI1 splice forms and the related MDS1/EVI1 (ME) gene, real-time quantitative polymerase chain reaction was performed in 534 untreated adults with de novo AML. EVI1-1D was highly expressed in 6% of cases (n = 32), whereas 7.8% were EVI1(+) (n = 41) when all splice variants were taken into account. High EVI1 predicted a distinctly worse event-free survival (HR = 1.9; P = .002) and disease-free survival (HR = 2.1, P = .006) following multivariate analysis. Importantly, we distinguished a subset of EVI1(+) cases that lacked expression of ME (EVI1(+)ME(-); n = 17) from cases that were ME(+) (EVI1(+)ME(+); n = 24). The atypical EVI1(+)ME(-) expression pattern exhibited cytogenetically detectable chromosomal 3q26 breakpoints in 8 cases. Fluorescence in situ hybridization revealed 7 more EVI1(+)ME(-) cases that carried cryptic 3q26 breakpoints, which were not found in the EVI1(+)ME(+) group. EVI1(+)ME(-) expression predicts an extremely poor prognosis distinguishable from the general EVI1(+) AML patients (overall survival [OS]: P < .001 and event-free survival [EFS]: P = .002). We argue that EVI1/ME quantitative expression analysis should be implemented in the molecular diagnostic procedures of AML.

ERCC1-XPF Endonuclease Facilitates DNA Double-Strand Break Repair

ABSTRACT ERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA lesions. However, mutations in ERCC1 or XPF in humans or mice cause a more severe phenotype than absence of NER, prompting a search for novel repair activities of the nuclease. In Saccharomyces cerevisiae, orthologs of ERCC1-XPF (Rad10-Rad1) participate in the repair of double-strand breaks (DSBs). Rad10-Rad1 contributes to two error-prone DSB repair pathways: microhomology-mediated end joining (a Ku86-independent mechanism) and single-strand annealing. To determine if ERCC1-XPF participates in DSB repair in mammals, mutant cells and mice were screened for sensitivity to gamma irradiation. ERCC1-XPF-deficient fibroblasts were hypersensitive to gamma irradiation, and γH2AX foci, a marker of DSBs, persisted in irradiated mutant cells, consistent with a defect in DSB repair. Mutant mice were also hypersensitive to irradiation, establishing an essential role for ERCC1-XPF in protecting against DSBs in vivo. Mice defective in both ERCC1-XPF and Ku86 were not viable. However, Ercc1−/−Ku86−/− fibroblasts were hypersensitive to gamma irradiation compared to single mutants and accumulated significantly greater chromosomal aberrations. Finally, in vitro repair of DSBs with 3′ overhangs led to large deletions in the absence of ERCC1-XPF. These data support the conclusion that, as in yeast, ERCC1-XPF facilitates DSB repair via an end-joining mechanism that is Ku86 independent.

Molecular cytogenetic and clinical findings in ETV6/ABL1-positive leukemia

Rearrangements of 12p, resulting from deletions or translocations, are common findings in hematologic malignancies. In many cases, these rearrangements target the ETV6 gene (previously called TEL) located at 12p13. Various partner genes have been implicated in the formation of fusion genes with ETV6. These include PDGFRB, JAK2, NTRK3, ABL2, and ABL1, each of which encodes for proteins with tyrosine kinase activity. To date, ETV6/ABL1 transcripts have been detected in only four patients with a leukemic disorder. Here, we describe one adult with chronic myeloid leukemia and a child with T‐cell acute lymphocytic leukemia with ETV6/ABL1. Molecular cytogenetic analysis confirmed that formation of an ETV6/ABL1 fusion in these patients required at least three chromosomal breaks and showed that each of these translocations is the result of a complex chromosomal rearrangement. Molecular analysis showed the presence of two fusion transcripts in both patients as the result of alternative splicing, questioning the suggested role of these transcripts in the lineage specificity. Clinical findings of these patients were compared to those of previously reported cases, and the possible clinical and biological similarities between ETV6/ABL1 and other fusion genes leading to increased tyrosine kinase activity are discussed.© 2000 Wiley‐Liss, Inc.

Identification of NUP98 abnormalities in acute leukemia: JARID1A (12p13) as a new partner gene

Chromosome rearrangements are found in many acute leukemias. As a result, genes at the breakpoints can be disrupted, forming fusion genes. One of the genes involved in several chromosome aberrations in hematological malignancies is NUP98 (11p15). As NUP98 is close to the 11p telomere, small translocations might easily be missed. Using a NUP98‐specific split‐signal fluorescence in situ hybridization (FISH) probe combination, we analyzed 84 patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia, or myelodysplastic syndrome with either normal karyotypes or 11p abnormalities to investigate whether there are unidentified 11p15 rearrangements. Neither NUP98 translocations nor deletions were identified in cases with normal karyotypes, indicating these aberrations may be very rare in this group. However, NUP98 deletions were observed in four cases with unbalanced 11p aberrations, indicating that the breakpoint is centromeric of NUP98. Rearrangements of NUP98 were identified in two patients, both showing 11p abnormalities in the diagnostic karyotype: a t(4;11)(q1?3;p15) with expression of the NUP98–RAP1GDS1 fusion product detected in a 60‐year‐old woman with AML‐M0, and an add(11)(p15) with a der(21)t(11;21)(p15;p13) observed cytogenetically in a 1‐year‐old boy with AML‐M7. JARID1A was identified as the fusion partner of NUP98 using 3′ RACE, RT‐PCR, and FISH. JARID1A, at 12p13, codes for retinoblastoma binding protein 2, a protein implicated in transcriptional regulation. This is the first report of JARID1A as a partner gene in leukemia.

Cytogenetic, Molecular Genetic and Pathological Analyses in 126 Meningiomas

Abstract. In a series of 126 meningiomas, tumor and patient charactersitics were investigated and statistically analyzed. A combined cytogenetic and molecular genetic approach was used to study chromosomal abnormalities and loss of markers on chromosome 22q. This apprach was successfully applied to 93 meningiomas. In 66 cases, complete or partial loss of chromosome 22 was observed and in at least 12 of them this chromosome was involved in structural aberrations. In addition to chromosome 22 changes, chromosomes 1, 6, 11, 13, 14, 18, 19, X, and Y were also frequently involved in structural and numerical aberrations. Statistical analysis revealed a significant association between the number of choromosomal abnormalities and tumor grade. Complex karyotypes predominated in the group of grade II/III meningiomas. Furthermore, other variables showed statistically (or marginally statistically) significant differences. Meningiomas from the convexity were more ofthen grade II/III, displayed predominantly (partial) loss of chromosome 22 and had complex karyotypes more often. These fetures were frequently found in meningiomas from males. Base meningiomas, on the other hand, occurred more often in females; they were usually grade I, showed loss of (parts of) chromosome 22 less often and displayed fewer additional chromosomal abnormalities.

Further characterization of the first seminoma cell line TCam-2

Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam‐2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q‐PCR) and NANOG, and the absence of CD30, SSX2‐4, and SOX2, confirms that TCam‐2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam‐2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam‐2 is the first well‐characterized seminoma‐derived cell line, with an exceptional mutation, rarely found in TGCTs.