ESMO recommendations on predictive biomarker testing for homologous recombination deficiency and PARP inhibitor benefit in ovarian cancer.

BACKGROUND: Homologous recombination repair deficiency (HRD) is a frequent feature of high-grade serous ovarian, fallopian tube and peritoneal carcinoma (HGSC) and is associated with sensitivity to PARP inhibitor (PARPi) therapy. HRD testing provides an opportunity to optimise PARPi use in HGSC but methodologies are diverse and clinical application remains controversial. MATERIALS AND METHODS: To define best practice for HRD testing in HGSC the ESMO Translational Research and Precision Medicine Working Group launched a collaborative project that incorporated a systematic review approach. The main aims were to (i) define the term 'HRD test'; (ii) provide an overview of the biological rationale and the level of evidence supporting currently available HRD tests; (iii) provide recommendations on the clinical utility of HRD tests in clinical management of HGSC. RESULTS: A broad range of repair genes, genomic scars, mutational signatures and functional assays are associated with a history of HRD. Currently, the clinical validity of HRD tests in ovarian cancer is best assessed, not in terms of biological HRD status per se, but in terms of PARPi benefit. Clinical trials evidence supports the use of BRCA mutation testing and two commercially available assays that also incorporate genomic instability for identifying subgroups of HGSCs that derive different magnitudes of benefit from PARPi therapy, albeit with some variation by clinical scenario. These tests can be used to inform treatment selection and scheduling but their use is limited by a failure to consistently identify a subgroup of patients who derive no benefit from PARPis in most studies. Existing tests lack negative predictive value and inadequately address the complex and dynamic nature of the HRD phenotype. CONCLUSIONS: Currently available HRD tests are useful for predicting likely magnitude of benefit from PARPis but better biomarkers are urgently needed to better identify current homologous recombination proficiency status and stratify HGSC management.

ESMO recommendations on predictive biomarker testing for homologous recombination deficiency and PARP inhibitor benefit in ovarian cancer

The European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group identified that there is currently uncertainty within the oncology community surrounding the different methods for HRD testing in HGSC. To address this, a collaborative project was launched with a number of clinicians and scientists with expertise in the fields of PARPi clinical trials, cancer genomics and DNA repair. The group defined three main aims for the project: (i) Define the term 'HRD test' and recommend how an HRD test's clinical validity is currently best assessed in the context of HGSC, (ii) provide an overview of the biological rationale and the level of evidence supporting currently available HRD tests, and (iii) provide recommendations on the clinical utility of HRD tests in clinical management of HGSC.

Cancer-associated fibroblasts as key regulators of the breast cancer tumor microenvironment.

Tumor cells exist in close proximity with non-malignant cells. Extensive and multilayered crosstalk between tumor cells and stromal cells tailors the tumor microenvironment (TME) to support survival, growth, and metastasis. Fibroblasts are one of the largest populations of non-malignant host cells that can be found within the TME of breast, pancreatic, and prostate tumors. Substantial scientific evidence has shown that these cancer-associated fibroblasts (CAFs) are not only associated with tumors by proximity but are also actively recruited to developing tumors where they can influence other cells of the TME as well as influencing tumor cell survival and metastasis. This review discusses the impact of CAFs on breast cancer biology and highlights their heterogeneity, origin and their role in tumor progression, ECM remodeling, therapy resistance, metastasis, and the challenges ahead of targeting CAFs to improve therapy response.

Mouse models in the era of large human tumour sequencing studies.

Cancer is a complex disease in which cells progressively accumulate mutations disrupting their cellular processes. A fraction of these mutations drive tumourigenesis by affecting oncogenes or tumour suppressor genes, but many mutations are passengers with no clear contribution to tumour development. The advancement of DNA and RNA sequencing technologies has enabled in-depth analysis of thousands of human tumours from various tissues to perform systematic characterization of their (epi)genomes and transcriptomes in order to identify (epi)genetic changes associated with cancer. Combined with considerable progress in algorithmic development, this expansion in scale has resulted in the identification of many cancer-associated mutations, genes and pathways that are considered to be potential drivers of tumour development. However, it remains challenging to systematically identify drivers affected by complex genomic rearrangements and drivers residing in non-coding regions of the genome or in complex amplicons or deletions of copy-number driven tumours. Furthermore, functional characterization is challenging in the human context due to the lack of genetically tractable experimental model systems in which the effects of mutations can be studied in the context of their tumour microenvironment. In this respect, mouse models of human cancer provide unique opportunities for pinpointing novel driver genes and their detailed characterization. In this review, we provide an overview of approaches for complementing human studies with data from mouse models. We also discuss state-of-the-art technological developments for cancer gene discovery and validation in mice.

RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer.

Background: BRCA1 and BRCA2 (BRCA1/2)-deficient tumors display impaired homologous recombination repair (HRR) and enhanced sensitivity to DNA damaging agents or to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi). Their efficacy in germline BRCA1/2 (gBRCA1/2)-mutated metastatic breast cancers has been recently confirmed in clinical trials. Numerous mechanisms of PARPi resistance have been described, whose clinical relevance in gBRCA-mutated breast cancer is unknown. This highlights the need to identify functional biomarkers to better predict PARPi sensitivity. Patients and methods: We investigated the in vivo mechanisms of PARPi resistance in gBRCA1 patient-derived tumor xenografts (PDXs) exhibiting differential response to PARPi. Analysis included exome sequencing and immunostaining of DNA damage response proteins to functionally evaluate HRR. Findings were validated in a retrospective sample set from gBRCA1/2-cancer patients treated with PARPi. Results: RAD51 nuclear foci, a surrogate marker of HRR functionality, were the only common feature in PDX and patient samples with primary or acquired PARPi resistance. Consistently, low RAD51 was associated with objective response to PARPi. Evaluation of the RAD51 biomarker in untreated tumors was feasible due to endogenous DNA damage. In PARPi-resistant gBRCA1 PDXs, genetic analysis found no in-frame secondary mutations, but BRCA1 hypomorphic proteins in 60% of the models, TP53BP1-loss in 20% and RAD51-amplification in one sample, none mutually exclusive. Conversely, one of three PARPi-resistant gBRCA2 tumors displayed BRCA2 restoration by exome sequencing. In PDXs, PARPi resistance could be reverted upon combination of a PARPi with an ataxia-telangiectasia mutated (ATM) inhibitor. Conclusion: Detection of RAD51 foci in gBRCA tumors correlates with PARPi resistance regardless of the underlying mechanism restoring HRR function. This is a promising biomarker to be used in the clinic to better select patients for PARPi therapy. Our study also supports the clinical development of PARPi combinations such as those with ATM inhibitors.

p53 deficiency induces cancer stem cell pool expansion in a mouse model of triple-negative breast tumors.

Triple-negative breast cancer is a heterogeneous disease characterized by the expression of basal cell markers, no estrogen or progesterone receptor expression and a lack of HER2 overexpression. Triple-negative tumors often display activated Wnt/ß-catenin signaling and most have impaired p53 function. We studied the interplay between p53 loss and Wnt/ß-catenin signaling in stem cell function and tumorigenesis, by deleting p53 from the mammary epithelium of K5ΔNßcat mice displaying a constitutive activation of Wnt/ß-catenin signaling in basal cells. K5ΔNßcat transgenic mice present amplification of the basal stem cell pool and develop triple-negative mammary carcinomas. The loss of p53 in K5ΔNßcat mice led to an early expansion of mammary stem/progenitor cells and accelerated the formation of triple-negative tumors. In particular, p53-deficient tumors expressed high levels of integrins and extracellular matrix components and were enriched in cancer stem cells. They also overexpressed the tyrosine kinase receptor Met, a feature characteristic of human triple-negative breast tumors. The inhibition of Met kinase activity impaired tumorsphere formation, demonstrating the requirement of Met signaling for cancer stem cell growth in this model. Human basal-like breast cancers with predicted mutated p53 status had higher levels of MET expression than tumors with wild-type p53. These results connect p53 loss and ß-catenin activation to stem cell regulation and tumorigenesis in triple-negative cancer and highlight the role of Met signaling in maintaining cancer stem cell properties, revealing new cues for targeted therapies.

Inhibition of the spindle assembly checkpoint kinase TTK enhances the efficacy of docetaxel in a triple-negative breast cancer model.

BACKGROUND: Triple-negative breast cancers (TNBC) are considered the most aggressive type of breast cancer, for which no targeted therapy exists at the moment. These tumors are characterized by having a high degree of chromosome instability and often overexpress the spindle assembly checkpoint kinase TTK. To explore the potential of TTK inhibition as a targeted therapy in TNBC, we developed a highly potent and selective small molecule inhibitor of TTK, NTRC 0066-0. RESULTS AND CONCLUSIONS: The compound is characterized by long residence time on the target and inhibits the proliferation of a wide variety of human cancer cell lines with potency in the same range as marketed cytotoxic agents. In cell lines and in mice, NTRC 0066-0 inhibits the phosphorylation of a TTK substrate and induces chromosome missegregation. NTRC 0066-0 inhibits tumor growth in MDA-MB-231 xenografts as a single agent after oral application. To address the effect of the inhibitor in breast cancer, we used a well-defined mouse model that spontaneously develops breast tumors that share key morphologic and molecular features with human TNBC. Our studies show that combination of NTRC 0066-0 with a therapeutic dose of docetaxel resulted in doubling of mouse survival and extended tumor remission, without toxicity. Furthermore, we observed that treatment efficacy is only achieved upon co-administration of the two compounds, which suggests a synergistic in vivo effect. Therefore, we propose TTK inhibition as a novel therapeutic target for neoadjuvant therapy in TNBC.

FOXA1 repression is associated with loss of BRCA1 and increased promoter methylation and chromatin silencing in breast cancer.

FOXA1 expression correlates with the breast cancer luminal subtype and patient survival. RNA and protein analysis of a panel of breast cancer cell lines revealed that BRCA1 deficiency is associated with the downregulation of FOXA1 expression. Knockdown of BRCA1 resulted in the downregulation of FOXA1 expression and enhancement of FOXA1 promoter methylation in MCF-7 breast cancer cells, whereas the reconstitution of BRCA1 in Brca1-deficent mouse mammary epithelial cells (MMECs) promoted Foxa1 expression and methylation. These data suggest that BRCA1 suppresses FOXA1 hypermethylation and silencing. Consistently, the treatment of MMECs with the DNA methylation inhibitor 5-aza-2'-deoxycitydine induced Foxa1 mRNA expression. Furthermore, treatment with GSK126, an inhibitor of EZH2 methyltransferase activity, induced FOXA1 expression in BRCA1-deficient but not in BRCA1-reconstituted MMECs. Likewise, the depletion of EZH2 by small interfering RNA enhanced FOXA1 mRNA expression. Chromatin immunoprecipitation (ChIP) analysis demonstrated that BRCA1, EZH2, DNA methyltransferases (DNMT)1/3a/3b and H3K27me3 are recruited to the endogenous FOXA1 promoter, further supporting the hypothesis that these proteins interact to modulate FOXA1 methylation and repression. Further co-immunoprecipitation and ChIP analysis showed that both BRCA1 and DNMT3b form complexes with EZH2 but not with each other, consistent with the notion that BRCA1 binds to EZH2 and negatively regulates its methyltransferase activity. We also found that EZH2 promotes and BRCA1 impairs the deposit of the gene silencing histone mark H3K27me3 on the FOXA1 promoter. These associations were validated in a familial breast cancer patient cohort. Integrated analysis of the global gene methylation and expression profiles of a set of 33 familial breast tumours revealed that FOXA1 promoter methylation is inversely correlated with the transcriptional expression of FOXA1 and that BRCA1 mutation breast cancer is significantly associated with FOXA1 methylation and downregulation of FOXA1 expression, providing physiological evidence to our findings that FOXA1 expression is regulated by methylation and chromatin silencing and that BRCA1 maintains FOXA1 expression through suppressing FOXA1 gene methylation in breast cancer.

Suppression of BRCA1 sensitizes cells to proteasome inhibitors.

BRCA1 is a multifunctional protein best known for its role in DNA repair and association with breast and ovarian cancers. To uncover novel biologically significant molecular functions of BRCA1, we tested a panel of 198 approved and experimental drugs to inhibit growth of MDA-MB-231 breast cancer cells depleted for BRCA1 by siRNA. 26S proteasome inhibitors bortezomib and carfilzomib emerged as a new class of selective BRCA1-targeting agents. The effect was confirmed in HeLa and U2OS cancer cell lines using two independent siRNAs, and in mouse embryonic stem (ES) cells with inducible deletion of Brca1. Bortezomib treatment did not cause any increase in nuclear foci containing phosphorylated histone H2AX, and knockdown of BRCA2 did not entail sensitivity to bortezomib, suggesting that the DNA repair function of BRCA1 may not be directly involved. We found that a toxic effect of bortezomib on BRCA1-depleted cells is mostly due to deregulated cell cycle checkpoints mediated by RB1-E2F pathway and 53BP1. Similar to BRCA1, depletion of RB1 also conferred sensitivity to bortezomib, whereas suppression of E2F1 or 53BP1 together with BRCA1 reduced induction of apoptosis after bortezomib treatment. A gene expression microarray study identified additional genes activated by bortezomib treatment only in the context of inactivation of BRCA1 including a critical involvement of the ERN1-mediated unfolded protein response. Our data indicate that BRCA1 has a novel molecular function affecting cell cycle checkpoints in a manner dependent on the 26S proteasome activity.

Mammary gland-specific ablation of focal adhesion kinase reduces the incidence of p53-mediated mammary tumour formation.

BACKGROUND: Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in spontaneous mammary tumour development caused by loss of tumour suppressor genes such as p53 is lacking. Alterations in the tumour suppressor gene p53 have been implicated in over 50% of human breast cancers. Given that elevated FAK expression highly correlates with p53 mutation status in human breast cancer, we set out to investigate the importance of FAK in p53-mediated spontaneous mammary tumour development. METHODS: To directly assess the role of FAK, we generated mice with conditional inactivation of FAK and p53. We generated female p53(lox/lox)/FAK(+/+)/WapCre, p53(lox/lox)/FAK(flox/+)/WapCre and p53(lox/lox)/FAK(flox/-)/WapCre mice, and mice with WapCre-mediated conditional expression of p53(R270H), the mouse equivalent of human p53(R273H) hot spot mutation, together with conditional deletion of FAK, P53(R270H/+)/FAK(lox/+)/WapCre and p53(R270H/+)/FAK(flox/-)/WapCre mice. All mice were subjected to one pregnancy to induce WapCre-mediated deletion of p53 or expression of p53 R270H, and Fak genes flanked by two loxP sites, and subsequently followed the development of mammary tumours. RESULTS: Using this approach, we show that FAK is important for p53-induced mammary tumour development. In addition, mice with the mammary gland-specific conditional expression of p53 point mutation R270H, the mouse equivalent to human R273H, in combination with conditional deletion of Fak showed reduced incidence of p53(R270H)-induced mammary tumours. In both models these effects of FAK were related to reduced proliferation in preneoplastic lesions in the mammary gland ductal structures. CONCLUSIONS: Mammary gland-specific ablation of FAK hampers p53-regulated spontaneous mammary tumour formation. Focal adhesion kinase deletion reduced proliferative capacity of p53 null and p53(R270H) mammary epithelial cells but did not lead to increased apoptosis in vivo. Our data identify FAK as an important regulator in mammary epithelial cell proliferation in p53-mediated and p53(R270H)-induced mammary tumour development.

Epidermal Snail expression drives skin cancer initiation and progression through enhanced cytoprotection, epidermal stem/progenitor cell expansion and enhanced metastatic potential.

Expression of the EMT-inducing transcription factor Snail is enhanced in different human cancers. To investigate the in vivo role of Snail during progression of epithelial cancer, we used a mouse model with skin-specific overexpression of Snail. Snail transgenic mice spontaneously developed distinct histological subtypes of skin cancer, such as basal cell carcinoma, squamous cell carcinoma and sebaceous gland carcinoma. Development of sebaceous gland carcinomas strongly correlated with the direct and complete repression of Blimp-1, a central regulator of sebocyte homeostasis. Snail expression in keratinocyte stem cells significantly promotes their proliferation associated with an activated FoxM1 gene expression signature, resulting in a larger pool of Mts24-marked progenitor cells. Furthermore, primary keratinocytes expressing Snail showed increased survival and strong resistance to genotoxic stress. Snail expression in a skin-specific p53-null background resulted in accelerated formation of spontaneous tumours and enhanced metastasis. Our data demonstrate that in vivo expression of Snail results in de novo epithelial carcinogenesis by allowing enhanced survival, expansion of the cancer stem cell pool with accumulated DNA damage, a block in terminal differentiation and increased proliferation rates of tumour-initiating cells.

Opportunities and hurdles in the treatment of BRCA1-related breast cancer.

BRCA1 functions as a classical tumor suppressor in breast and ovarian cancer. While the role of BRCA1 in homology-directed repair of DNA double-strand breaks contributes to its tumor suppressive activity, it also renders BRCA1-deficient cells highly sensitive to DNA-damaging agents. Although BRCA1 deficiency is therefore considered to be an attractive therapeutic target, re-activation of BRCA1 by secondary mutations has been shown to cause therapy resistance. In this review, we will assess the role of BRCA1 in both hereditary and sporadic breast cancer and discuss how different functionalities of the BRCA1 protein can contribute to its tumor suppressor function. In addition, we will discuss how this knowledge on BRCA1 function can help to overcome the hurdles encountered in the clinic and improve current treatment strategies for patients with BRCA1-related breast cancer.

Frat oncoproteins act at the crossroad of canonical and noncanonical Wnt-signaling pathways.

Wnt-signal transduction is critical for development and tissue homeostasis in a wide range of animal species and is frequently deregulated in human cancers. Members of the Frat/GBP family of glycogen synthase kinase 3beta (Gsk3b)-binding oncoproteins are recognized as potent activators of the Wnt/beta-catenin pathway in vertebrates. Here, we reveal a novel, Gsk3b-independent function of Frat converging on the activation of JNK and AP-1. Both these have been used as readouts for the noncanonical Frizzled/PCP pathway, which controls polarized cell movements and the establishment of tissue polarity. We find that Frat synergizes with Diversin, the mammalian homolog of the Drosophila PCP protein diego, in the activation of JNK/AP-1 signaling. Importantly, Frat mutants deficient for binding to Gsk3b retain oncogenic activity in vivo, suggesting that Wnt/beta-catenin-independent events contribute to Frat-induced malignant transformation. The observed activities of Frat are reminiscent of the dual function of Dishevelled in the Wnt/beta-catenin and Frizzled/PCP pathways and suggest that Frat may also function to bridge canonical and noncanonical Wnt pathways.

Crisis intervention and acute psychiatry in Amsterdam, 20 years of change: a historical comparison of consultations in 1983 and 2004-2005.

AIM: There has been a striking increase in the number of compulsory admission proceedings in the Netherlands since 1992, to such an extent that treatment in Amsterdam's psychiatric clinics is in danger of being dominated by coercive treatment. Our aim was to establish a picture of the changes in emergency psychiatry that have contributed to the increase in the number of acute compulsory admissions. METHODS: A cohort (N = 460) of psychiatric emergency consultations with the city crisis service in 1983 was compared with a similar cohort (N = 436) in 2004-2005. The study focused on the following variables: patient characteristics, crisis-service procedures and consultation outcomes. RESULTS: Compared with 1983, there are now more services involved in crisis support in the public domain for psychiatric patients. The number of patients referred by the police has risen from 29% to 63%. In 1983, all consultations took place where the patients were located; at present, 60% take place at the crisis service premises. The number of psychotic patients in the cohort has increased from 52.0% and 63.3 %. There has been an increase in the proportion of compulsory admissions and a sharp decrease in the proportion of voluntary admissions from 61% to 28% of all admissions. Overall, the percentage of consultations leading to a psychiatric admission has fallen from 42% to 27%. CONCLUSION: The front-line outreach service of 1983 has changed into a specialist psychiatric emergency department with a less pronounced outreach component. Voluntary admissions to psychiatric hospitals have almost disappeared as a feature of the crisis service.

Preclinical mouse models for BRCA1-associated breast cancer.

A substantial part of all hereditary breast cancer cases is caused by BRCA1 germline mutations. In this review, we will discuss the insights into BRCA1 functions that we obtained from mouse models with conventional and conditional mutations in Brca1. The most advanced models closely resemble human BRCA1-related breast cancer and may therefore be useful for addressing clinically relevant questions.

[Changes in crisis intervention and emergency psychiatry, Amsterdam emergency consultations in 1983 and 2005]. / Veranderingen in crisisinterventie en acute psychiatrie; Amsterdamse consulten in 1983 en 2005.

BACKGROUND: Since 1992 The Netherlands has seen a striking increase in the number of compulsory admissions. There is a danger that coercion will become the dominant form of treatment in the Amsterdam clinics. AIM: To build up a picture of the changes in emergency psychiatry which have contributed to the increase in the number of acute compulsory admissions. METHOD: A cohort (n = 460) of consultations conducted by the city crisis service in 1983 was compared with a similar cohort (n = 436) of consultations conducted in 2004-2005 based on the following variables: crisis-service procedures, patient characteristics, diagnosis and consultation outcomes. RESULTS: In 2003-2004 more services were involved with psychiatric patients in acute situations in the public domain than were involved in 1983. The number of patients referred by the police almost doubled, rising from 29.3 to 62.7%. In 1983 all consultations took place where the patients were located; in 2004-2005 60% took place at the premises of the crisis service. The number of psychotic patients in the cohort rose from 52 to 63.3%. There was a rise in the number of compulsory admissions (from 16.7 to 20%) and a sharp decline in voluntary admissions (from 25.7 to 7.6%). The total number of admissions following a consultation decreased from 42 to 28%. CONCLUSIONS: The front-line outreach service of 1983 has been transformed into a specialist psychiatric emergency department with only a modest outreach component. Voluntary admissions via the consultation service have almost ceased. Further research is needed into the characteristics of the consultations and into the variables that play a role in the use of compulsion in emergency psychiatry.

Mouse models of BRCA1 and BRCA2 deficiency: past lessons, current understanding and future prospects.

Germline mutations in BRCA1 and BRCA2 are responsible for a large proportion of hereditary breast and ovarian cancers. Soon after the identification of both genes in the mid-1990s, investigators set out to develop mouse models for the associated disease. Whereas conventional Brca1 and Brca2 mouse mutants did not reveal a strong phenotype in a heterozygous setting, most homozygous mutations caused embryonic lethality. Consequently, development of mouse models for BRCA-associated tumorigenesis required the generation of tissue-specific conditional knockout animals. In this review, we give an overview of the conventional and the conditional mouse models of BRCA1 and BRCA2 deficiency generated over the last decade, as well as the contribution of these models to our understanding of the biological and molecular functions of BRCA1 and BRCA2. The most advanced mouse models for BRCA1- and BRCA2-associated tumorigenesis mimic human disease to the extent that they can be used in studies addressing clinically relevant questions. These models will help to resolve yet unanswered questions and to translate our increasing knowledge of BRCA1 and BRCA2 biology into clinical practice.

Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer.

Inheritance of one defective BRCA2 allele predisposes humans to breast cancer. To establish a mouse model for BRCA2-associated breast cancer, we generated mouse conditional mutants with BRCA2 and/or p53 inactivated in various epithelial tissues, including mammary-gland epithelium. Although no tumors arose in mice carrying conditional Brca2 alleles, mammary and skin tumors developed frequently in females carrying conditional Brca2 and Trp53 alleles. The presence of one wildtype Brca2 allele resulted in a markedly delayed tumor formation; loss of the wildtype Brca2 allele occurred in a subset of these tumors. Our results show that inactivation of BRCA2 and of p53 combine to mediate mammary tumorigenesis, and indicate that disruption of the p53 pathway is pivotal in BRCA2-associated breast cancer.

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-ER(T) 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.

A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent.

Conditional gene inactivation using the Cre/loxP system is widely used, but the difficulty in properly regulating Cre expression remains one of the bottlenecks. One approach to regulate Cre activity utilizes a mutant estrogen hormone-binding domain (ERT) to keep Cre inactive unless the non-steroidal estrogen analog 4-hydroxytamoxifen (OHT) is present. Here we describe a mouse strain expressing Cre-ERT from the ubiquitously expressed ROSA26 (R26) locus. We demonstrate efficient temporal and spatial regulation of Cre recombination in vivo and in primary cells derived from these mice. We show the existence of marked differences in recombination frequencies between different substrates within the same cell. This has important consequences when concurrent switching of multiple alleles within the same cell is needed, and highlights one of the difficulties that may be encountered when using reporter mice as indicator strains.