http://repub.eur.nl/res/aut/4574/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/32551/ 2012-06-13T00:00:00Z The ultimate purpose of sexual reproduction, which depends on specialized male and female anatomy and physiology, is to enable continuation of a species and introduction of genetic diversity. In mammals the developmental path towards a male or a female is in principle determined at the moment of fertilization, when either a Y- or an X-chromosome is inherited from the father. The subsequent chromosomal constitution, either XY (male) or XX (female) (referred to as chromosomal sex), will eventually drive formation of a testis or an ovary (the so called gonadal sex). This in turn will result in the next step in sex determination (the phenotypic sex), ultimately leading to a phenotypical male or female respectively. Because of the relevance of the general principles related to this phenomenon in understanding the various levels in which pathological gonadal processes can occur, the next paragraphs will explain these issues in more detail. These are schematically shown in Figure 1 and 2. Some of the items to be discussed are (partially) presented in Chapter 3. http://repub.eur.nl/res/pub/37965/ 2012-03-01T00:00:00Z Disorders of sex development (DSD) are congenital conditions where chromosomal, gonad or genital development is atypical. In a significant proportion of 46,XY DSD cases it is not possible to identify a causative mutation, making genetic counseling difficult and potentially hindering optimal treatment. Here, we describe the analysis of a 46,XY DSD patient that presented at birth with ambiguous genitalia. Histological analysis of the surgically removed gonads showed bilateral undifferentiated gonadal tissue and immature testis, both containing malignant germ cells. We screened genomic DNA from this patient for deletions and duplications using an Illumina whole-genome SNP microarray. This analysis revealed a heterozygous deletion within the WWOX gene on chromosome 16, removing exons 6-8. Analysis of parental DNA showed that the deletion was inherited from the mother. cDNA analysis confirmed that the deletion maintained the reading frame, with exon 5 being spliced directly onto exon 9. This deletion is the first description of a germline rearrangement affecting the coding sequence of WWOX in humans. Previously described Wwox knockout mouse models showed gonadal abnormalities, supporting a role for WWOX in human gonad development. http://repub.eur.nl/res/pub/38248/ 2012-02-03T00:00:00Z Disorders of sex development (DSD) are defined as a congenital condition in which development of chromosomal, gonadal or anatomical sex is atypical. DSD patients with gonadal dysgenesis or hypovirilization, containing part of the Y chromosome (GBY), have an increased risk for malignant type II germ cell tumors (GCTs: seminomas and nonseminomas). DSD may be diagnosed in newborns (e.g., ambiguous genitalia), or later in life, even at or after puberty. Here we describe three independent male patients with a GCT; two were retrospectively recognized as DSD, based on the histological identification of both carcinoma in situ and gonadoblastoma in a single gonad as the cancer precursor. Hypospadias and cryptorchidism in their history are consistent with this conclusion. The power of recognition of these parameters is demonstrated by the third patient, in which the precursor lesion was diagnosed before progression to invasiveness. Early recognition based on these clinical parameters could have prevented development of (metastatic) cancer, to be treated by systemic therapy. All three patients showed a normal male 46,XY karyotype, without obvious genetic rearrangements by high-resolution whole-genome copy number analysis. These cases demonstrate overlap between DSD and the so-called testicular dysgenesis syndrome (TDS), of significant relevance for identification of individuals at increased risk for development of a malignant GCT. Copyright http://repub.eur.nl/res/pub/34997/ 2012-01-13T00:00:00Z Disorder of sex development (DSD) patients in Indonesia most often do not receive a proper diagnostic evaluation and treatment. This study intended to categorize 88 Indonesian patients in accordance with the new consensus DSD algorithm. Diagnostic evaluation including clinical, hormonal, genetic, imaging, surgical, and histological parameters was performed. Fifty-three patients were raised as males, and 34 as females. Of 22 patients with 46, XX DSD, 15 had congenital adrenal hyperplasia, while in one patient, an ovarian Leydig cell tumor was found. In all 58 46, XY DSD patients, 29 were suspected of a disorder of androgen action (12 with an androgen receptor mutation), and in 9, gonadal dysgenesis was found and, in 20, severe hypospadias e.c.i. Implementation of the current consensus statement in a resource-poor environment is very difficult. The aim of the diagnostic workup in developing countries should be to end up with an evidence-based diagnosis. This is essential to improve treatment and thereby to improve the patients' quality of life. http://repub.eur.nl/res/pub/22975/ 2010-12-01T00:00:00Z Mammalian parthenogenesis could not survive but aborted during mid-gestation, presumably because of lack of paternal gene expression. To understand the molecular mechanisms underlying the failure of parthenogenesis at early stages of development, we performed global gene expression profiling and functional analysis of parthenogenetic blastocysts in comparison with those of blastocysts from normally fertilized embryos. Parthenogenetic blastocysts exhibited changes in the expression of 749 genes, of which 214 had lower expression and 535 showed higher expressions than fertilized embryos using a minimal 1.8-fold change as a cutoff. Genes important for placenta development were decreased in their expression in parthenote blastocysts. Some maternally expressed genes were up-regulated and paternal-related genes were down-regulated. Moreover, aberrantly increased Wnt signaling and reduced mitogen-activated protein kinase (MAPK) signaling were associated with early parthenogenesis. The protein level of extracellular signal-regulated kinase 2 (ERK2) was low in parthenogenetic blastocysts compared with that of fertilized blastocysts 120 h after fertilization. 6-Bromoindirubin-3′-oxime, a specific glycogen synthase kinase-3 (GSK-3) inhibitor, significantly decreased embryo hatching. The expression of several imprinted genes was altered in parthenote blastocysts. Gene expression also linked reduced expression of Xist to activation of X chromosome. Our findings suggest that failed X inactivation, aberrant imprinting, decreased ERK/MAPK signaling and possibly elevated Wnt signaling, and reduced expression of genes for placental development collectively may contribute to abnormal placenta formation and failed fetal development in parthenogenetic embryos. http://repub.eur.nl/res/pub/28631/ 2010-09-01T00:00:00Z Certain patients with disorders of sex development (DSD), who bear Y chromosome material in their karyotype, are at increased risk for the development of type II germ cell tumors (GCT), which arise from early fetal germ cells. DSD gonads frequently harbor immature germ cells which express early fetal germ cell markers. Some of them (e.g. OCT3/4 and NANOG) seem to be of pathogenetic relevance in GCT development providing cells with the ability of pluripotency, proliferation and apoptosis suppression. Also TSPY (testis-specific protein Y-encoded), the main candidate for the so-called gonadoblastoma locus on Y chromosome, is overexpressed in germ cells of DSD patients and possibly contributes to their survival and proliferation. Nowadays, the use of immunohistochemical methods is highly relevant in identifying DSD gonads at risk. The risk for GCT development varies. While the prevalence of GCT is 15% in patients with partial androgen insensitivity, it may reach more than 30% in patients with gonadal dysgenesis. Patients with complete androgen insensitivity and ovotesticular DSD develop malignancies in 0.8% and 2.6% of cases, respectively. However, these data may be biased for various reasons. To better estimate the risk in individual groups of DSD, further investigations on large patient series are needed. Copyright http://repub.eur.nl/res/pub/28586/ 2010-04-01T00:00:00Z Disorders of sex development (DSD), previously referred to as intersex, has been recognised as one of the main risk factors for development of type II germ cell tumours (GCTs), that is, seminomas/dysgerminomas and non-seminomas (e.g., embryonal carcinoma, yolk sac tumour, choriocarcinoma and teratoma). Within the testis, this type of cancer is the most frequent malignancy in adolescent and young adult Caucasian males. Although these males are not known to have dysgenetic gonads, the similarities in the resulting tumours suggest a common aetiological mechanism(s), -genetically, environmentally or a combination of both. Within the group of DSD patients, being in fact congenital conditions, the risk of malignant transformation of germ cells is highly heterogeneous, depending on a number of parameters, some of which have only recently been identified. Understanding of these recent insights will stimulate further research, with the final aim to develop an informative clinical decision tree for DSD patients, which includes optimal (early) diagnosis without overtreatment, such as prophylactic gonadectomy in the case of a low tumour risk. http://repub.eur.nl/res/pub/24551/ 2009-06-12T00:00:00Z Patients with disorders of sex development (DSD), especially those with gonadal dysgenesis and hypovirilization, are at risk of developing the so-called type II germ cell tumors (GCTs). Both carcinoma in situ and gonadoblastoma (GB) can be the precursor lesion, resulting in a seminomatous or non-seminomatous invasive cancer. SRY mutations residing in the HMG domain are found in 10-15% of 46,XY gonadal dysgenesis cases. This domain contains two nuclear localization signals (NLSs). In this study, we report a unique case of a phenotypical normal woman, diagnosed as a patient with 46,XY gonadal dysgenesis, with an NLS missense mutation, on the basis of the histological diagnosis of a unilateral GB. The normal role of SRY in gonadal development is the upregulation of SOX9 expression. The premalignant lesion of the initially removed gonad was positive for OCT3/4, TSPY and stem cell factor in germ cells, and for FOXL2 in the stromal component (ie, granulosa cells), but not for SOX9. On the basis of these findings, prophylactical gonadectomy of the other gonad was performed, also showing a GB lesion positive for both FOXL2 (ovary) and SOX9 (testis). The identified W70L mutation in the SRY gene resulted in a 50% reduction in the nuclear accumulation of the mutant protein compared with wild type. This likely explains the diminished SOX9 expression, and therefore the lack of proper Sertoli cell differentiation during development. This case shows the value of the proper diagnosis of human GCTs in identification of patients with DSD, which allows subsequent early diagnosis and prevention of the development of an invasive cancer, likely to be treated by chemotherapy at young age. http://repub.eur.nl/res/pub/16256/ 2009-03-01T00:00:00Z Testicular germ cell tumors of adolescents and adults (TGCTs; the so-called type II variant) are the most frequent malignancies found in Caucasian males between 20 and 40 years of age. The incidence has increased over the last decades. TGCTs are divided into seminomas and nonseminomas, the latter consisting of the subgroups embryonal carcinoma, yolk-sac tumor, teratoma, and choriocarcinoma. The pathogene-sis starts in utero, involving primordial germ cells/gonocytes that are blocked in their differentiation, and develops via the precursor lesion carcinoma in situ toward invasiveness. TGCTs are totipotent and can be considered as stem cell tumors. The developmental capacity of their cell of origin, the primordial germ cells/gonocyte, is demonstrated by the different tumor histologies of the invasive TGCTs. Seminoma represents the germ cell lineage, and embryonal carcinoma is the undifferentiated component, being the stem cell population of the nonseminomas. Somatic differentiation is seen in the teratomas (all lineages), whereas yolk-sac tumors and choriocarcinoma represent extra-embryonal differentiation. Seminomas are highly sensitive to irradiation and (DNA damaging) chemotherapy, whereas most nonseminomatous elements are less susceptible to radiation, although still sensitive to chemotherapy, with the exception of teratoma. To allow early diagnosis and follow up, appropriate markers are mandatory to discriminate between the different subgroups. In this review, a summary will be given related to several recent developments in TGCT research, especially selected because of their putative clinical impact. http://repub.eur.nl/res/pub/29703/ 2008-09-10T00:00:00Z Disorders of sex development (DSD), previously known as intersex, refer to congenital conditions in which development of chromosomal, gonadal, or anatomical sex is atypical. Patients with specific variants of this disorder have an elevated risk for the development of so-called type II germ cell cancers, i.e., the seminomatous and nonseminatous tumors, referred to as germ cell tumors (GCTs). Specifically DSD patients with gonadal dysgenesis or hypovirilization are at risk. A prerequisite for type II GCT formation is the presence of a specific part of the Y chromosome (referred to as the GBY region), with the TSPY gene being the most likely candidate. Also the octamer binding transcription factor OCT3/4 is consistently expressed in all type II GCTs with pluripotent potential, as well as in the precursor lesions carcinoma in situ (CIS) in case of a testis and gonadoblastoma (GB) in the DSD gonad. The actual risk for malignant transformation in individual DSD patients is hard to predict, because of confusing terminology referring to the different forms of DSD, and unclear criteria for identification of the presence of malignant germ cells, especially in young patients. This is specifically due to the phenomenon of delay of germ cell maturation, which might result in over diagnosis. This review will give novel insight into the pathogenesis of the type II GCTs through the study of patients with various forms of DSD for which the underlying molecular defect is known. To allow optimal understanding of the pathogenesis of this type of cancers, first normal gonadal development, especially regarding the germ cell lineage, will be discussed, after which type II GCTs will be introduced. Subsequently, the relationship between type II GCTs and DSD will be described, resulting in a number of new insights into the development of the precursor lesions of these tumors. http://repub.eur.nl/res/pub/28917/ 2008-05-01T00:00:00Z The transcription factors SOX9 and FOXL2 are required for male and female mammalian gonadal development. We have used specific antibodies to investigate the role of these key proteins in disorders of sex development (DSD), specifically inter-sex states. In normal gonads, SOX9 was found to be restricted to the presence of (pre-)Sertoli cells, while FOXL2 was found in granulosa cells, and in stromal cells interpreted as early ovarian stroma. Both proteins were found within a single patient, when testicular and ovarian development was present; and within the same gonad, when both differentiation lineages were identified, as in ovotesticular DSD (ie hermaphrodite). Especially SOX9 was informative to support the presence of early testicular development (ie seminiferous tubules), expected based on morphological criteria only. In a limited number of DSD cases, FOXL2 was found within reasonably well-developed seminiferous tubules, but double staining demonstrated that it was never strongly co-expressed with SOX9 in the same cell. All seminiferous tubules containing carcinoma in situ (CIS), the malignant counterpart of a primordial germ cell, ie the precursor of type II germ cell tumours of the testis, seminomas and non-seminomas, showed the presence of SOX9 and not FOXL2. In contrast, gonadoblastomas (GBs), the precursor of the same type of cancer, in a dysgenetic gonad, showed expression of FOXL2 and no, or only very low, SOX9 expression. These findings indicate that gonadal differentiation, ie testicular or ovarian, determines the morphology of the precursor of type II germ cell tumours, CIS or GB, respectively. We show that in DSD patients, the formation of either ovarian or/and testicular development can be visualized using FOXL2 and SOX9 expression, respectively. In addition, it initiates a novel way to study the role of the supportive cells in the development of either CIS or GB. Copyright http://repub.eur.nl/res/pub/30047/ 2008-03-01T00:00:00Z 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. http://repub.eur.nl/res/pub/35122/ 2007-11-01T00:00:00Z Testicular germ cell tumours (GCTs) of adolescents and adults can be subdivided into seminomas (referred to as dysgerminomas of the ovary) and non-seminomas, all referred to as type II GCTs. They originate from carcinoma in situ (CIS), being the malignant counterparts of primordial germ cells (PGCs)/gonocytes. The invasive components mimic embryogenesis, including the stem cell component embryonal carcinoma (EC), the somatic lineage teratoma (TE), and the extra-embryonic tissues yolk sac tumour (YST) and choriocarcinoma (CH). The other type is the so-called spermatocytic seminomas (SS, type III GCT), composed of neoplastic primary spermatocytes. We reported previously that the miRNAs hsa-miR 371-373 cluster is involved in overruling cellular senescence induced by oncogenic stress, allowing cells to become malignant. Here we report the first high-throughput screen of 156 microRNAs in a series of type II and III GCTs (n = 69, in duplicate) using a quantitative PCR-based approach. After normalization to allow intersample analysis, the technical replicates clustered together, and the previous hsa-miRNA 371-373 cluster finding was confirmed. Unsupervised cluster analysis demonstrated that the cell lines are different from the in vivo samples. The in vivo samples, both normal and malignant, clustered predominantly based on their maturation status. This parallels normal embryogenesis, rather than chromosomal anomalies in the tumours. miRNAs within a single cluster showed a similar expression pattern, implying common regulatory mechanisms. Normal testicular tissue expressed most discriminating miRNAs at a higher level than SE and SS. Moreover, differentiated non-seminomas showed overexpression of discriminating miRNAs. These results support the model that miRNAs are involved in regulating differentiation of stem cells, retained in GCTs. Copyright http://repub.eur.nl/res/pub/36583/ 2007-10-01T00:00:00Z Malignant mesothelioma (MM) is an asbestos-induced tumor that acquires aneuploid DNA content during the tumorigenic process. We used instable MM cell lines as an in vitro model to study the impact of DNA copy-number changes on gene expression profiling, in the course of their chromosomal redistribution process. Two MM cell lines, PMR-MM2 (early passages of in vitro culture) and PMR-MM7 (both early and late passages of in vitro culture), were cytogenetically characterized. Genomic gains and losses were precisely defined using microarray-based comparative genomic hybridization (array-CGH), and minimal overlapping analysis led to the identification of the common unbalanced genomic regions. Using the UI33Plus 2.0 Affymetrix gene chip array, we analyzed PMR-MM7 early and late passages for genome-wide gene expression, and correlated the differentially expressed genes with copy-number changes. The presence of a high number of genetic imbalances occurring from early to late culture steps reflected the tendency of MM cells toward genomic instability. The selection of specific chromosomal abnormalities observed during subsequent cultures demonstrated the spontaneous evolution of the cancer cells in an in vitro environment. MM cell lines were characterized by copy-number changes associated with the TP53 apoptotic pathway already present at the first steps of in vitro culture. Prolonged culture led to acquisition of additional chromosomal copy-number changes associated with dysregulation of genes involved in cell adhesion, regulation of mitotic cell cycle, signal transduction, carbohydrate metabolism, motor activity, glycosaminoglycan biosynthesis, protein binding activity, lipid transport, ATP synthesis, and methyltransferase activity. http://repub.eur.nl/res/pub/36972/ 2007-09-01T00:00:00Z Disorders of sex development (DSD), previously referred to as intersex disorders, comprise a variety of anomalies defined by congenital conditions in which chromosomal, gonadal, or anatomical sex is atypical. Besides issues such as gender assignment, clinical and diagnostic evaluation, surgical and psychosocial management, and sex steroid replacement, the significantly increased risk for developing specific types of malignancies is both clinically and biologically relevant. This relates to germ-cell tumors specifically in DSD patients with hypovirilization or gonadal dysgenesis. The presence of a well-defined part of the Y chromosome (known as the GBY region) is a prerequisite for malignant transformation, for which the testis-specific protein on the Y chromosome (TSPY) is a likely candidate gene. The precursor lesions of these cancers are carcinoma in situ (CIS)/intratubular germ-cell neoplasia unclassified (ITGCNU) in testicular tissue and gonadoblastoma in those without obvious testicular differentiation. Most recently, undifferentiated gonadal tissue (UGT) has been identified as the likely precursor for gonadoblastoma. The availability of markers for the different developmental stages of germ cells allows detailed investigation of the characteristics of normal and (pre)malignant germ cells. Although informative in a diagnostic setting for adult male patients, these markers - such as OCT3/4 - cannot easily distinguish (pre)malignant germ cells from germ cells showing delayed maturation. This latter phenomenon is frequently found in gonads of DSD patients, and may be related to the risk of malignant transformation. Thus, the mere application of these markers might result in over-diagnosis and unnecessary gonadectomy. It is proposed that morphological and histological evaluation of gonadal tissue, in combination with OCT3/4 and TSPY double immunohistochemistry and clinical parameters, is most informative in estimating the risk for germ-cell tumor development in the individual patient, and might in future be used to develop a decision tree for optimal management of patients with DSD. http://repub.eur.nl/res/pub/8364/ 2005-01-01T00:00:00Z The androgen receptor (AR) is essential for development of the male gender and in the growth of the majority of prostate cancers. Agonists as well as most antagonists induce translocation of the receptor to the nucleus, whereas only agonists can activate AR function. Antagonists are therefore used in the therapy of metastasized prostate cancer. To obtain insight into the mechanism by which antagonists block AR function in living cells, we studied nuclear mobility and localization of green fluorescent protein (GFP)-tagged AR in the presence of either the agonist R1881 or the antagonists bicalutamide and hydroxyflutamide. As controls we investigated a non-DNA-binding AR mutant (A573D) and two mutants (W741C and T877A) with broadened ligand specificity. We demonstrate that in the presence of R1881, AR localizes in numerous intranuclear foci and, using complementary fluorescence recovery after photobleaching (FRAP) approaches and computer modelling, that a fraction of AR ( approximately 10-15%) is transiently immobilized in a DNA-binding-dependent manner (individual ARs being immobile for approximately 45 seconds). By contrast, antagonist-bound GFP-AR showed no detectable immobile fraction and the mobility was similar to that of the R1881-liganded non-DNA-binding mutant (A573D), indicating that antagonists do not induce the relatively stable DNA-binding-dependent immobilization observed with agonist-bound AR. Moreover, in the presence of bicalutamide and hydroxyflutamide GFP-AR was homogeneously distributed in the nucleus. Binding of bicalutamide and hydroxyflutamide to GFP-AR(W741C) and GFP-AR(T877A), respectively, resulted in similar mobility and heterogeneous nuclear distribution as observed for R1881-liganded GFP-AR. The live cell studies indicate that the investigated antagonists interfere with events early in the transactivation function of the AR. http://repub.eur.nl/res/pub/13416/ 2004-09-01T00:00:00Z Among nuclear receptors, the androgen receptor (AR) is unique in that its ligand-binding domain (LBD) interacts with the FXXLF motif in the N-terminal domain, resembling coactivator LXXLL motifs. We compared AR- and estrogen receptor alpha-LBD interactions of the wild-type AR FXXLF motif and coactivator transcriptional intermediary factor 2 LXXLL motifs and variants of these motifs. Random mutagenesis revealed a key role for the F residues in FXXLF motifs in high-affinity and selective AR LBD interaction. The FXXLF motif in full-length AR and transcriptional intermediary factor 2 LXXLL motifs competed for an overlapping binding site. A computer model of the AR LBD/AR FXXLF complex showed that the bulky F residues are buried in a deep coactivator-binding groove. The corresponding groove in estrogen receptor alpha LBD is considerably shallower, explaining lack of binding of any of the FXXLF motifs tested. FXXLF and LXXLL motif interaction depended on different charged amino acid residues in the AR LBD present at opposite ends of the coactivator groove. In conclusion, our data demonstrate the importance of a deep hydrophobic groove and alternative usage of charged amino acids in specifying peptide binding to the AR LBD. http://repub.eur.nl/res/pub/10020/ 2002-01-01T00:00:00Z The N-terminal domain (NTD) and the ligand-binding domain (LBD) of the androgen receptor (AR) exhibit a ligand-dependent interaction (N/C interaction). Amino acids 3-36 in the NTD (AR3-36) play a dominant role in this interaction. Previously, it has been shown that a PhixxPhiPhi motif in AR3-36, 23FxxLF27, is essential for LBD interaction. We demonstrate in the current study that AR3-36 can be subdivided into two functionally distinct fragments: AR3-13 and AR16-36. AR3-13 does not directly interact with the AR LBD, but rather contributes to the transactivation function of the AR.NTD-AR.LBD complex. AR16-36, encompassing the 23FxxLF27 motif, is predicted to fold into a long amphipathic alpha-helix. A second PhixxPhiPhi candidate protein interaction motif within the helical structure, 30VREVI34, shows no affinity to the LBD. Within AR16-36, amino acid residues in and flanking the 23FxxLF27 motif are demonstrated to modulate N/C interaction. Substitution of Q24 and N25 by alanine residues enhances N/C interaction. Substitution of amino acids flanking the 23FxxLF27 motif by alanines are inhibitory to LBD interaction. http://repub.eur.nl/res/pub/3162/ 1999-05-20T00:00:00Z We have generated mice deficient in O6-methylguanine DNA methyltransferase activity encoded by the murine Mgmt gene using homologous recombination to delete the region encoding the Mgmt active site cysteine. Tissues from Mgmt null mice displayed very low O6-methylguanine DNA methyltransferase activity, suggesting that Mgmt constitutes the major, if not the only, O6-methylguanine DNA methyltransferase. Primary mouse embryo fibroblasts and bone marrow cells from Mgmt -/- mice were significantly more sensitive to the toxic effects of the chemotherapeutic alkylating agents 1,3-bis(2-chloroethyl)-1-nitrosourea, streptozotocin and temozolomide than those from Mgmt wild-type mice. As expected, Mgmt-deficient fibroblasts and bone marrow cells were not sensitive to UV light or to the crosslinking agent mitomycin C. In addition, the 50% lethal doses for Mgmt -/- mice were 2- to 10-fold lower than those for Mgmt +/+ mice for 1,3-bis(2chloroethyl)-1-nitrosourea, N-methyl-N-nitrosourea and streptozotocin; similar 50% lethal doses were observed for mitomycin C. Necropsies of both wild-type and Mgmt -/mice following drug treatment revealed histological evidence of significant ablation of hematopoietic tissues, but such ablation occurred at much lower doses for the Mgmt -/- mice. These results demonstrate the critical importance of O6-methylguanine DNA methyltransferase in protecting cells and animals against the toxic effects of alkylating agents used for cancer chemotherapy.