Mammalian MutS homologue 5 is required for chromosome pairing in meiosis.

MSH5 (MutS homologue 5) is a member of a family of proteins known to be involved in DNA mismatch repair. Germline mutations in MSH2, MLH1 and GTBP (also known as MSH6) cause hereditary non-polyposis colon cancer (HNPCC) or Lynch syndrome. Inactivation of Msh2, Mlh1, Gtmbp (also known as Msh6) or Pms2 in mice leads to hereditary predisposition to intestinal and other cancers. Early studies in yeast revealed a role for some of these proteins, including Msh5, in meiosis. Gene targeting studies in mice confirmed roles for Mlh1 and Pms2 in mammalian meiosis. To assess the role of Msh5 in mammals, we generated and characterized mice with a null mutation in Msh5. Msh5-/- mice are viable but sterile. Meiosis in these mice is affected due to the disruption of chromosome pairing in prophase I. We found that this meiotic failure leads to a diminution in testicular size and a complete loss of ovarian structures. Our results show that normal Msh5 function is essential for meiotic progression and, in females, gonadal maintenance.

MiR-205-driven downregulation of cholesterol biosynthesis through SQLE-inhibition identifies therapeutic vulnerability in aggressive prostate cancer.

Prostate cancer (PCa) shows strong dependence on the androgen receptor (AR) pathway. Here, we show that squalene epoxidase (SQLE), an enzyme of the cholesterol biosynthesis pathway, is overexpressed in advanced PCa and its expression correlates with poor survival. SQLE expression is controlled by micro-RNA 205 (miR-205), which is significantly downregulated in advanced PCa. Restoration of miR-205 expression or competitive inhibition of SQLE led to inhibition of de novo cholesterol biosynthesis. Furthermore, SQLE was essential for proliferation of AR-positive PCa cell lines, including abiraterone or enzalutamide resistant derivatives, and blocked transactivation of the AR pathway. Inhibition of SQLE with the FDA approved antifungal drug terbinafine also efficiently blocked orthotopic tumour growth in mice. Finally, terbinafine reduced levels of prostate specific antigen (PSA) in three out of four late-stage PCa patients. These results highlight SQLE as a therapeutic target for the treatment of advanced PCa.

Somatic hypermutation in MutS homologue (MSH)3-, MSH6-, and MSH3/MSH6-deficient mice reveals a role for the MSH2-MSH6 heterodimer in modulating the base substitution pattern.

Although the primary function of the DNA mismatch repair (MMR) system is to identify and correct base mismatches that have been erroneously introduced during DNA replication, recent studies have further implicated several MMR components in somatic hypermutation of immunoglobulin (Ig) genes. We studied the immune response in mice deficient in MutS homologue (MSH)3 and MSH6, two mutually exclusive partners of MSH2 that have not been examined previously for their role in Ig hypermutation. In Msh6(-)/- and Msh3(-)/-/Msh6(-)/- mice, base substitutions are preferentially targeted to G and C nucleotides and to an RGYW hot spot, as has been shown previously in Msh2(-)/- mice. In contrast, Msh3(-)/- mice show no differences from their littermate controls. These findings indicate that the MSH2-MSH6 heterodimer, but not the MSH2-MSH3 complex, is responsible for modulating Ig hypermutation.

The interleukin 2 receptor alpha chain/CD25 promoter is a target for nuclear factor of activated T cells.

The expression of the murine interleukin (IL)-2 receptor alpha chain/CD25 is strongly induced at the transcriptional level after T cell activation. We show here that nuclear factor of activated T cell (NF-AT) factors are involved in the control of CD25 promoter induction in T cells. NF-ATp and NF-ATc bind to two sites around positions -585 and -650 located upstream of the proximal CD25 promoter. Immediately 3' from these NF-AT motifs, nonconsensus sites are located for the binding of AP-1-like factors. Mutations of sites that suppress NF-AT binding impair the induction and strong NF-ATp-mediated transactivation of the CD25 promoter in T cells. In T lymphocytes from NF-ATp-deficient mice, the expression of CD25 is severely impaired, leading to a delayed IL-2 receptor expression after T cell receptor (TCR)/CD3 stimulation. Our data indicate an important role for NF-AT in the faithful expression of high affinity IL-2 receptors and a close link between the TCR-mediated induction of IL-2 and IL-2 receptor alpha chain promoters, both of which are regulated by NF-AT factors.

Mdm2-SNP309 polymorphism in prostate cancer: no evidence for association with increased risk or histopathological tumour characteristics.

The search for inherited cancer susceptibility factors is a major focus of epidemiologic cancer studies. Analyses of single-nucleotide polymorphisms (SNP) in a variety of genes revealed a correlation between a specific allele variant and cancer predisposition. Human mouse double-minute 2 protein (Mdm2) is a cellular E3 ligase capable of ubiquitination and degradation of p53. Therefore, Mdm2 is a crucial factor of cell cycle control and cell survival. The Mdm2 promoter SNP309 was shown to increase Mdm2 expression and can, thereby, inhibit the p53 pathway. This SNP was found to be associated with increased risk and early onset of various malignancies. For prostate cancer no studies are reported to date. In a case-control study we determined the distribution of the Mdm2 SNP309 in 145 male subjects with prostate cancer and in 124 male controls without any malignancy using RFLP analysis. Cases and controls showed a similar distribution of the SNP (P=0.299). Genotype distribution showed neither an association with histopathological characteristics of the tumours nor with prognosis. Age at disease onset was also not modified by the SNP. This first study of the Mdm2 SNP309 in prostate cancer patients suggests no correlation between a certain allelic variant and an increased cancer risk.

The survival impact of neoadjuvant hormonal therapy before radical prostatectomy for treatment of high-risk prostate cancer.

BACKGROUND: Several randomized controlled trials assessed the outcomes of patients treated with neoadjuvant hormonal therapy (NHT) before radical prostatectomy (RP). The majority of them included mainly low and intermediate risk prostate cancer (PCa) without specifically assessing PCa-related death (PCRD). Thus, there is a lack of knowledge regarding a possible effect of NHT on PCRD in the high-risk PCa population. We aimed to analyze the effect of NHT on PCRD in a multicenter high-risk PCa population treated with RP, using a propensity-score adjustment. METHODS: This is a retrospective multi-institutional study including patients with high-risk PCa defined as: clinical stage T3-4, PSA >20 ng ml-1 or biopsy Gleason score 8-10. We compared PCRD between RP and NHT+RP using competing risks analysis. Correction for group differences was performed by propensity-score adjustment. RESULTS: After application of the inclusion/exclusion criteria, 1573 patients remained for analysis; 1170 patients received RP and 403 NHT+RP. Median follow-up was 56 months (interquartile range 29-88). Eighty-six patients died of PCa and 106 of other causes. NHT decreased the risk of PCRD (hazard ratio (HR) 0.5; 95% confidence interval (CI) 0.32-0.80; P=0.0014). An interaction effect between NHT and radiotherapy (RT) was observed (HR 0.3; 95% CI 0.21-0.43; P<0.0008). More specifically, of patients who received adjuvant RT, those who underwent NHT+RP had decreased PCRD rates (2.3% at 5 year) compared to RP (7.5% at 5 year). The retrospective design and lack of specific information about NHT are possible limitations. CONCLUSIONS: In this propensity-score adjusted analysis from a large high-risk PCa population, NHT before surgery significantly decreased PCRD. This effect appeared to be mainly driven by the early addition of RT post-surgery. The specific sequence of NHT+RP and adjuvant RT merits further study in the high-risk PCa population.

Tumorigenesis in Mlh1 and Mlh1/Apc1638N mutant mice.

An3 1 KAL I MutL homologue 1 (MLH1) is a member of the family of proteins required for DNA mismatch repair. Germ-line mutations in MLH1 lead to the cancer susceptibility syndrome hereditary nonpolyposis colorectal cancer (HNPCC). We generated mice carrying a null mutation in the Mlh1 gene. We showed that mice heterozygous and homozygous for the Mlh1 gene are predisposed to developing tumors of the gastrointestinal (GI) tract, lymphomas, and a number of other tumor types. We also examined the role of adenomatous polyposis coli gene (Apc) gene mutations in the GI tumors of Mlh1 mutant mice by different methods and showed that the GI tumors in Mlh1 mice express little or no adenomatous polyposis coli protein. When an Apc gene mutation was bred into the Mlh1 mutant mice, the GI tumor incidence increased 40-100-fold. The wild-type Apc allele in these tumors was found to contain mutations. Together, these results show that we have developed two mouse models for human HNPCC and that the mechanisms of tumor development in the GI tract of these mice involve loss of Apc gene function in a manner very similar to that seen in the GI tumors of HNPCC.

The DNA mismatch repair genes Msh3 and Msh6 cooperate in intestinal tumor suppression.

Repair of mismatches in DNA in mammalian cells is mediated by a complex of proteins that are members of two highly conserved families of genes referred to as MutS and MutL homologues. Germline mutations in several members of these families, MSH2, MSH6, MLH1, and PMS2, but not MSH3, are responsible for hereditary non-polyposis colorectal cancer. To examine the role of MSH3, we generated a mouse with a null mutation in this gene. Cells from Msh3-/- mice are defective in repair of insertion/ deletion mismatches but can repair base-base mismatches. Msh3-/- mice develop tumors at a late age. When the Msh3-/- and Msh6-/- mutations are combined, the tumor predisposition phenotype is indistinguishable from Msh2-/- or Mlh1-/- mice. These results suggest that MSH3 cooperates with MSH6 in tumor suppression.

Predicting prostate cancer-specific outcome after radical prostatectomy among men with very high-risk cT3b/4 PCa: a multi-institutional outcome study of 266 patients.

BACKGROUND: The value of radical prostatectomy (RP) as an approach for very high-risk prostate cancer (PCa) patients is controversial. To examine the risk of 10-year cancer-specific mortality (CSM) and other-cause mortality (OCM) according to clinical and pathological characteristics of very high-risk cT3b/4 PCa patients treated with RP as the primary treatment option. METHODS: In a multi-institutional cohort, 266 patients with very high-risk cT3b/4 PCa treated with RP were identified. All patients underwent RP and pelvic lymph-node dissection. Competing-risk analyses assessed 10-year CSM and OCM before and after stratification for age and Charlson comorbidity index (CCI). RESULTS: Overall, 34 (13%) patients died from PCa and 73 (28%) from OCM. Ten-year CSM and OCM rates ranged from 5.6% to 12.9% and from 10% to 38%, respectively. OCM was the leading cause of death in all subgroups. Age and comorbidities were the main determinants of OCM. In healthy men, CSM rate did not differ among age groups (10-year CSM rate for ⩽64, 65-69 and ⩾70 years: 16.2%, 11.5% and 17.1%, respectively). Men with a CCI ⩾1 showed a very low risk of CSM irrespective of age (10-year CSM: 5.6-6.1%), whereas the 10-year OCM rates increased with age up to 38% in men ⩾70 years. CONCLUSION: Very high-risk cT3b/4 PCa represents a heterogeneous group. We revealed overall low CSM rates despite the highly unfavorable clinical disease. For healthy men, CSM was independent of age, supporting RP even for older men. Conversely, less healthy patients had the highest risk of dying from OCM while sharing very low risk of CSM, indicating that this group might not benefit from an aggressive surgical treatment. Outcome after RP as the primary treatment option in cT3b/4 PCa patients is related to age and comorbidity status.

Inefficient termination of antigen responses in NF-ATp-deficient mice.

In order to elucidate the role of NF-ATp, one of the most prominent members of family of NF-AT transcription factors in peripheral T lymphocytes, in T cell activation and differentiation we created NF-ATp-deficient mice by gene targeting. Such NF-ATp-/- mice are born and appear to develop a normal immune system. Apart from clear-cut defects in the synthesis of mRNAs for Th2-type lymphokines, such as IL-4, IL-5, IL-10 and IL-13, in primary and secondary stimulations of spleen cells in vitro, of a distinct impaired deletion of V beta 11+/CD4+ T lymphocytes from these mice was detected after superantigen injection. Moreover, NF-ATp-/- mice older than 6 weeks show an 2-5 fold increase in number of lymphocytes. This is correlated with an increased expression of activation markers CD44 and CD69 and decreased expression of CD62.

In vitro skewing of TCR transgenic CD4+ T cells from interleukin-2 deficient mice towards Th1 and Th2 in the absence of exogenous interleukin-2.

Previous experiments from several groups have indicated that in vitro priming for Th2 cells rigorously requires IL-4 but also depends on IL-2 [1-3]. On the other hand, IL-2 deficient mice characteristically have highly increased serum levels of the Th2-dependent isotypes IgG1 and IgE [4]. The overproduction of these isotypes is lost in IL-2 x IL-4 double deficient animals [5]. To readdress the question of a need for IL-2 for Th2 skewing in vitro we used T cells from IL-2-/- mice also transgenic for the DO11.10 TCR which is specific for OVA + IAd [6]. CD4+ cells from these mice were primed in vitro on IL-2-/- dendritic cells in the presence of OVA peptide and IL-4, IL-12 and IL-15, respectively. Following restimulation, cytokine production was analysed by intracellular staining with anti IL-4 and anti IFNgamma antibodies and flow cytometry. The data show that IL-4 primes IL-2-/- T cells for IL-4 production even in the absence of exogenous IL-2, while IL-12, as expected, polarises towards IFNgamma production. The ability to be primed for IL-4 production in the absence of IL-2 was also exhibited by naive CD4+CD62LlowTCR transgenic IL-2-/- cells and thus was not restricted to the CD44high CD62Llow cells which make up a high proportion of CD4+ cells in IL-2 deficient mice. We conclude that IL-2 is not absolutely required for in vitro skewing of naive T cells towards Th2.

STAT3 and SMAD1 signaling in Medaka embryonic stem-like cells and blastula embryos.

The activation and transcriptional activity of signal transducer and activator of transcription 3 (STAT3) is essential for maintaining mouse embryonic stem (ES) cell cultures in an undifferentiated state. However, reports from human and monkey ES-cell culture suggest that STAT3 is dispensable for pluripotency in these systems. At the same time, BMP signaling via smad1 was shown to be able to counteract STAT3 signaling in murine ES-cell cultures, while it influences differentiation in multifaceted ways in other cellular contexts. Hence, the question arises whether the signaling situation found in mice or primates and human ES-cells represent the rule or the exception. With this study, we want to contribute an answer to this question from an evolutionary perspective. Therefore, we analyzed the expression and activation status of the Medaka (Oryzias latipes) STAT3 and SMAD1 in Medaka ES-cell-like cultures and their in vivo counterpart, the Medaka blastula embryo. While SMAD signaling is active in the culture system as well as in blastula embryos, our results indicate that STAT3 is inactive and can thus not be involved in pluripotency control of blastula cells or their derived pluripotent in vitro counterparts. These results suggest that the signaling pathways active in the mouse ES-cell culture system represent the exception, while inactivity of STAT3 is apparently the rule in vertebrate ES-cell cultures.

Normal clonal expansion but impaired Fas-mediated cell death and anergy induction in interleukin-2-deficient mice.

Despite a normal development of all major lymphoid subsets, with time, interleukin-2 (IL-2)-deficient mice develop a fatal immunopathology. The disease phenotype is characterized by lymphoadenopathy, splenomegaly, T cell infiltration of various organs, overproduction of a number of cytokines and autoantibody formation. Phenotypically, CD4+ and CD8+ T cells exhibit features characteristic of antigenically experienced cells. The accumulation of cells with a memory phenotype together with the previous suggestion of an involvement of IL-2 in the termination phase of immune responses prompted us to study the fate of superantigen-reactive T cells in IL-2-deficient mice in comparison to their IL-2-producing littermates. We show that expansion in vivo of CD4+ and, to a lesser extent, CD8+ T cells reactive to the superantigens staphylococcal enterotoxin A and B (SEA and SEB) proceeds normally in the absence of IL-2, but that fewer CD4+ cells are subsequently deleted. The residual superantigen-reactive cells fail to become anergic as measured by proliferation in vitro in response to the same superantigen. T cell blasts generated in vitro from lymph node cells of IL-2-deficient mice by superantigen stimulation in the absence of exogenous IL-2 also fail to become anergic. In contrast to cells from IL-2-producing littermates, they do not exhibit Fas-induced apoptosis when cultured on anti-Fas antibody-coated plates, although Fas expression by IL-2-deficient cells is normal or even elevated compared to the IL-2-producing control cells. The data suggest that activation of T cells in the absence of IL-2 fails to generate a signal which is necessary to activate the apoptotic pathway and thus leads to an accumulation of antigen-experienced cells and the chronic inflammatory responses observed in IL-2-deficient mice.

[The spectrum of lymphoproliferations and malignant lymphoma after organ transplant]. / Das Spektrum lymphoproliferativer Erkrankungen und maligner Lymphome nach Organtransplantation.

Lymphoproliferative disease in patients with organ transplantation has increasingly been diagnosed in recent years. It is caused by immunosuppression after organ transplantion. A large percentage of these tumors is associated with Epstein-Barr virus infection. The classification differentiates between early lesions, polymorphic and monomorphic lymphoproliferations. Whereas the early lesions are often polyclonal, the remaining groups are mostly monoclonal. Polymorphic lymphoproliferative disease shows a wide spectrum of B-cell differentiation, whereas monomorphic proliferations resemble sproadic malignant lymphomas. The latter also show genetic abberations of the c-myc-, Ras-, p53- and Bcl6-genes. With reference to the germinal center reaction, posttransplant lymphoproliferations have to be assigned to the post-follicular activated B-cell type. While until 1991 only 2% of the cases of PTLD had an EBV-association, the EBV-associated PTLD have been frequently observed in recent times and presently constitute about 20% of the cases. EBV-negative PTLD constitute a negative prognostic factor. Posttransplant lymphomas might also represent a model for sporadic lymphoproliferative disease, that for example also occurs at elderly patients.

Cyclosporin A interferes with the inducible degradation of NF-kappa B inhibitors, but not with the processing of p105/NF-kappa B1 in T cells.

The transcription factor NF-kappa B controls the induction of numerous cytokine promoters during the activation of T lymphocytes. Inhibition of T cell activation by the immunosuppressants cyclosporin A (CsA) and FK506 exerts a suppressive effect on the induction of these NF-kappa B-controlled cytokine promoters. We show for human Jurkat T leukemia cells, as well as human and mouse primary T lymphocytes, that this inhibitory effect is accompanied by an impaired nuclear translocation of the Rel proteins c-Rel, RelA/p65 and NF-kappa B1/p50, whereas the nuclear appearance of RelB remains unaffected. CsA does not interfere with the synthesis of Rel proteins, but prevents the inducible degradation of cytosolic NF-kappa B inhibitors I kappa B alpha and I kappa B beta upon T cell activation. CsA neither inhibits the processing of the NF-kappa B1 precursor p105 to p50, nor does it "stabilize" the C-terminal portion of p105, I kappa B gamma, which is degraded during p105 processing to mature p50. These results indicate that CsA interferes with a specific event in the signal-induced degradation of I kappa B alpha and I kappa B beta, but does not affect the processing of NF-kappa B1/p105 to p50.

RelA/p65 is a molecular target for the immunosuppressive action of protein kinase A.

Stimulation of the protein kinase A (PKA) signalling pathway exerts an inhibitory effect on the proliferation of numerous cells, including T lymphocytes. In CD4+ T helper cells, stimulation of PKA leads to suppression of interleukin 2 (IL-2) induction, while induction of the genes coding for the lymphokines IL-4 and IL-5 is enhanced. We show that the differential effect of PKA activity on induction of the IL-2 and IL-4 genes is mediated through their promoters. One major target of the suppressive effect of PKA is the kappa B site in the IL-2 promoter. A kappa B site is missing in the IL-4 promoter. Mutations preventing factor binding to the IL-2 kappa B site result in a loss of PKA-mediated suppression of IL-2 promoter activity. Furthermore, activation of the PKA signalling pathway impairs the inducible activity of multiple kappa B sites of the IL-2 promoter, but not of other factor binding sites. The reduction in activity of kappa B sites in activated and PKA-stimulated T cells is accompanied by changes in the concentration and DNA binding of Rel/NF-kappa B factors. Stimulation of the PKA pathway in Jurkat T cells with the PKA activator forskolin leads to an increase in synthesis of c-Rel and p105/p50, while synthesis of p65/RelA remains unchanged. However, nuclear translocation and DNA binding of p65 is distinctly impaired, probably due to a retarded degradation of I kappa B-alpha. In a similar way, stimulation of the PKA signalling pathway inhibits nuclear translocation of p65 and generation of nuclear kappa B complexes in peripheral T lymphocytes from murine lymph nodes. These results indicate that PKA-mediated suppression of NF-kappa B activity plays an important role in the control of activation of peripheral T lymphocytes.